Salts of diphosphate phosphoramidate of nucleosides as anticancer compounds

ABSTRACT

The present invention relates to compounds comprising a salt of a diphosphate phosphoramidate of a nucleoside drug, e.g., clofarabine. The compounds are useful in the treatment of cancer, e.g., leukemia.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/769,635, filed on Jun. 4, 2020, which is a § 371 national stageapplication based on Patent Cooperation Treaty Application serial numberPCT/GB2018/053535, filed on Dec. 5, 2018, which claims the benefit ofpriority to GB 1720279.7, filed Dec. 5, 2017. The entirety of each ofthese applications is incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The present invention provides compounds useful in the treatment ofcancer, e.g., leukaemia. The compounds comprise a salt of a diphosphatephosphoramidate. The present invention also provides formulations ofsaid compounds and uses of said compounds.

BACKGROUND TO THE INVENTION

Nucleoside based drugs have become a powerful tool in the treatment ofhuman disease. In the treatment of cancer, in particular, the use ofnucleoside drugs such as gemcitabine, clofarabine, cytarabine,fludarabine is widespread.

The effectiveness of all nucleoside drugs however can be limited by bothinherent and acquired resistance mechanisms.

One way in which the efficacy of nucleoside drugs can be improved is byadministration as a drug of the ProTide class. Drugs of the ProTideclass are prodrugs of monophosphorylated nucleosides and have been shownto be particularly potent therapeutic agents in the fields of bothantivirals and oncology. These compounds appear to avoid many of theresistance mechanisms which limit the utility of the parent nucleosides(see, for example, ‘Application of ProTide Technology to Gemcitabine: ASuccessful Approach to Overcome the Key Cancer Resistance MechanismsLeads to a New Agent (NUC-1031) in Clinical Development’; Slusarczyk etal; J. Med. Chem.; 2014, 57, 1531-1542; McGuigan et al.; PhosphoramidateProTides of the anticancer agent FUDR successfully deliver the preformedbioactive monophosphate in cells and confer advantage over the parentnucleoside; J. Med. Chem.; 2011, 54, 7247-7258; and Vande Voorde et al.;The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5fluoro-2′-deoxyuridine, is independent of activation by thymidine kinaseand insensitive to degradation by phosphorolytic enzymes; Biochem.Pharmacol.; 2011, 82, 441-452; WO2005/012327; WO2006/100439;WO2012/117246 and WO2016/083830). An exemplary ProTide is NUC-1031, aProTide of gemcitabine:

Whilst the ProTide strategy has proved to be very effective for certainnucleosides, it is not as effective with other nucleoside drugs. Thereis therefore a need to find further methods of potentiating nucleosidedrug molecules.

Drugs of the ProTide class can also be poorly soluble in aqueoussolvents and this can make administration challenging.

It is an aim of certain embodiments of the present invention to providea therapeutic agent that, in use, has therapeutic efficacy in theprophylaxis or treatment of cancer.

It is an aim of certain embodiments of the present invention to providea therapeutic agent that, in use, has a greater therapeutic efficacy inthe prophylaxis or treatment of cancer than the parent nucleotide or thecorresponding ProTide.

It is an aim of certain embodiments of the present invention to providea therapeutic agent that is more conveniently administered than thecorresponding ProTide.

Certain embodiments of the present invention solve some or all of theabove stated objects.

STATEMENT OF THE INVENTION

In a first aspect of the invention there is provided a compound offormula (I), or a pharmaceutically acceptable salt thereof:

whereinR¹ is independently at each occurrence selected from: C₁-C₂₄-alkyl,C₃-C₂₄-alkenyl, C₃-C₂₄-alkynyl, C₀-C₄-alkylene-C₃-C₈-cycloalkyl andC₀-C₄-alkylene-aryl;R² and R³ are each independently at each occurrence selected from H,C₁-C₆-alkyl and C₁-C₃-alkylene-R⁷; or R² and R³ together with the atomto which they are attached form a 3- to 6-membered cycloalkyl orheterocycloalkyl group;R⁴ is independently at each occurrence H or C₁-C₄-alkyl;or R⁴, a group selected from R² and R³ and the atoms to which they areattached may form a 3- to 6-membered heterocycloalkyl group;R⁵ is independently at each occurrence selected from aryl, 5-, 6-, 9- or10-membered heteroaryl, C₃-C₈-cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₁-C₃-alkylene-R^(5a) and C₁-C₈-alkyl; said aryl beingoptionally fused to C₆-C₈-cycloalkyl;R^(5a) is independently at each occurrence selected from aryl, 5-, 6-,9- or 10-membered heteroaryl, C₃-C₈-cycloalkyl, 3- to 7-memberedheterocycloalkyl, said aryl being optionally fused to C₆-C₈-cycloalkyl;R⁶ is independently selected from:

R⁷ is independently at each occurrence selected from aryl, imidazole,indole, SR^(a), OR^(a), CO₂R^(a), CO₂NR^(a)R^(a), NR^(a)R^(b) andNH(═NH)NH₂;R⁸ is independently selected from H and

Z¹ and Z² are each independently selected from O and S;Y is independently selected from H, F, Cl and OMe;X is independently at each occurrence a pharmaceutically acceptablecation;wherein any aryl group is either phenyl or naphthyl;wherein where any of R¹, R², R³, R⁴, R⁵ or R⁷ is an alkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl, that alkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl group is optionally substitutedwith from 1 to 4 substituents selected from: halo, nitro, cyano,NR^(a)R^(a), NR^(a)S(O)₂R^(a), NR^(a)C(O)R^(a), NR^(a)CONR^(a)R^(a),NR^(a)CO₂R^(a), OR^(a); SR^(a), SOR^(a), SO₃R^(a), SO₂R^(a),SO₂NR^(a)R^(a), CO₂R^(a), C(O)R^(a), CONR^(a)R^(a),CR^(a)R^(a)NR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl andC₁-C₄-haloalkyl; wherein RE is independently at each occurrence selectedfrom: H and C₁-C₄-alkyl; and R^(b) is independently at each occurrenceselected from: H, C₁-C₄-alkyl and C(O)—C₁-C₄-alkyl.

The inventors have found that phosphoramidated diphosphates of formula(I) have good activities against a range of cancer cell lines, includingsolid tumour and haematological cancer cell lines. In many cases thediphosphates of formula (I) are more active than the correspondingProTide.

Diphosphate prodrugs reported in the literature such as for examplenucleoside pyrophosphate diesters based on glicerides (K. Y. Hostetleret al. J. Biol. Chem. 1990, 265, 6112 6117; G. M. T. can Wijk, K.Y.Hostetler et al. Biochim. Biophys. Acta Lipids Lipid Metab. 1991,1084, 307-310) are known to be unstable due to cleavage of a bond of theP—O—P group, thus releasing the corresponding nucleoside monophosphateinstead of desired diphosphate. The inventors have found, however, thatdeprotonation of the α-phosphate group of diphosphate phosphoramidatesprovides high stability. The ionic nature of the compounds of theinvention also means that they are more soluble in water than thecorresponding ProTide.

A number of enzymatic processes are needed to convert a ProTide into thecorresponding monophosphate nucleotide, including ester cleavage bycarboxylesterases and cleavage of the resultant amino acid from themonophosphate nucleoside phosphoramidase-type enzymes. The diphosphatephosphoramidates of the invention are larger molecules, having adifferent shape and they are ionic, casting doubt over whether they willinteract with the relevant enzymes in the same way for conversion to thediphosphate nucleotide. Indeed, in preliminary data, the inventors haveshown that the diphosphate phosphoramidates of formula (I) are stablewith respect to carboxypeptidases, for example, carboxypeptidase Y, theenzymes that carry out the ester cleavage step in the conversion ofdrugs of the ProTide class to monophosphates. Nevertheless, thediphosphate phosphoramidates of formula (I) are still active in cells,suggesting that an alternative mechanism of action is taking placecompared to drugs of the ProTide class. The inventors have also shownthat where there is an alkyloxy group on the phosphoramidate phosphorous(OR^(S) in Formula I) the compounds of the invention are active. Indrugs of the ProTide class, this is not typically the case and onlycompounds having aryloxy groups at this position provide good activity.This is understood to be because the aryloxy group acts as a leavinggroup during the activation of drugs of the ProTide class. Again, thisindicates that the diphosphate phosphoramidates of the invention areactivated via a different mechanism to drugs of the ProTide class.

In embodiments, the compound of formula (I) is a compound of formula(II):

Diphosphate phosphoramidates of clofarabine such as those of formula(II) were more active than the corresponding ProTide against a number ofcancer cell lines, including the haematological cell lines.

In embodiments, the compound of formula (I) is a compound of formula(III):

In embodiments, the compound of formula (I) is a compound of formula(IV):

In embodiments, the compound of formula (I) is a compound of formula(V):

In embodiments, the compound of formula (I) is a compound of formula(VI):

In embodiments, the compound of formula (I) is a compound of formula(VII):

In these embodiments, it may be that R¹, R², R³, R⁵ and X are the sameat both occurrences. PGP-30,C 1

The following statements apply to compounds of any of formulae (I) to(VII). These statements are independent and interchangeable. In otherwords, any of the features described in any one of the followingstatements may (where chemically allowable) be combined with thefeatures described in one or more other statements below. In particular,where a compound is exemplified or illustrated in this specification,any two or more of the statements below which describe a feature of thatcompound, expressed at any level of generality, may be combined so as torepresent subject matter which is contemplated as forming part of thedisclosure of this invention in this specification.

R¹ may be independently at each occurrence selected from: C₁-C₂₄-alkyl,C₃-C₂₄-alkenyl, C₃-C₂₄-alkynyl, C₀-C₄-alkylene-C₃-C₆-cycloalkyl andC₀-C₄-alkylene-aryl.

R¹ may be independently at each occurrence selected from: C₁-C₂₄-alkyl,C₀-C₄-alkylene-C₃-C₈-cycloalkyl and CH₂-aryl. R¹ may be independently ateach occurrence selected from: C₁-C₁₀-alkyl, C₄-C₆-cycloalkyl andbenzyl. R¹ may be independently at each occurrence selected from:C₁-C₈-alkyl, C₆-cycloalkyl and benzyl.

R¹ may be C₁-C₈ alkyl.

R¹ may be selected such that it comprises three or more carbon atoms. R¹may be selected such that it comprises five or more carbon atoms. R¹ maytherefore be selected such that it includes six or more carbon atoms. R¹is preferably selected such that it comprises only carbon and hydrogenatoms. R¹ may be selected from C₅-C₇-cycloalkyl, C₅-C₈-alkyl and benzyl,optionally wherein said groups are unsubstituted. R¹ may beunsubstituted benzyl. R¹ may be neopentyl. R¹ may be ethyl.

It may be that R³ is H. It may be that R³ is C₁-C₄-alkyl. It may be thatR³ is methyl. It may be that R² is selected from C₁-C₆-alkyl andC₁-C₃-alkylene-R⁷. It may be that R² is C₁-C₄-alkyl. It may be that R²is selected from methyl and isopropyl. R² may be methyl. R² may be H.

R⁴ is preferably H.

It may be that R⁴, a group selected from R² and R³, and the atoms towhich they are attached form a 3- to 6-membered heterocycloalkyl group.It may be that R⁴, a group selected from R² and R³, and the atoms towhich they are attached do not form a 3- to 6-membered heterocycloalkylgroup. It may be that R² and R³ are each independently at eachoccurrence selected from H, C₁-C₆-alkyl and C₁-C₃-alkylene-R⁷; or R² andR³ together with the atom to which they are attached form a 3- to6-membered heterocycloalkyl group; and R⁴ is independently at eachoccurrence H or C₁-C₄-alkyl.

It may be that R⁵ is substituted or unsubstituted phenyl, which may beoptionally fused to a C₆-C₈-cycloalkyl ring, e.g., a cyclohexane ring.It may be that R⁵ is substituted or unsubstituted phenyl. It may be thatR⁵ is substituted or unsubstituted naphthyl (e.g., 1-naphthyl).Preferably, R⁵ is selected from unsubstituted phenyl or unsubstitutednaphthyl (e.g., 1-naphthyl). Thus, R⁵ may be unsubstituted phenyl.Alternatively, R⁵ may be unsubstituted naphthyl (e.g., 1-naphthyl).

R⁶ may be

R⁶ may be

R⁶ may be

R⁶ may be

Y may be H. Y may be F.

R⁸ may be H. R⁸may be

Z² may be S. Z² may be O.

Where R⁸ is

it may be that R¹, R², R³, R⁵, X and Z^(Z) are the same at bothoccurrences.

R⁶ may be

R⁶ may be

R⁶ may be

The compound of formula (I) may be selected from:

X⁺ may be a metal cation or it may be an ammonium cation. X⁺ may be ametal cation, e.g., a cation of an alkali or alkali earth metal. X⁺ maybe an ammonium cation, e.g., a trialkylammonium cation or an ammoniumcation of a nitrogen heterocycle. Illustrative cations include thosederived from aluminium, arginine, benzathine, calcium, choline,diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine,potassium, sodium, tromethamine, triethylamine and zinc cations. X⁺ maybe a triethylamine cation.

Compounds of the invention comprise a chiral centre at the phosphorusatom (β-phosphorus) that is bonded to OR⁵. The compound may be presentas a mixture of phosphate diastereoisomers, as the (S)-epimer at thephosphorus atom in substantially diastereomerically pure form or as the(R)-epimer at the phosphorus atom in substantially diastereomericallypure form. ‘Substantially diastereomerically pure’ is defined for thepurposes of this invention as a diastereomeric purity of greater thanabout 90%. If present as a substantially diastereoisomerically pureform, the compound may have a diastereoisomeric purity of greater than95%, 98%, 99%, or even 99.5%. Alternatively, the compound may be presentas a mixture of phosphate diastereoisomers.

The (R)- and/or (S)-epimers of the compound can be obtained insubstantially diastereomerically pure form by chromatography, e.g., HPLCoptionally using a chiral column. Alternatively, the (R)- and/or(S)-epimers of the compound can be obtained in substantiallydiastereomerically pure form by crystallisation from an appropriatesolvent or solvent system.

According to a second aspect of the present invention, there is provideda compound of formula (I), or a pharmaceutically acceptable saltthereof, for use in a method of treatment.

According to a third aspect of the present invention, there is provideda compound of formula (I), or a pharmaceutically acceptable saltthereof, for use in the prophylaxis or treatment of cancer.

According to a fourth aspect of the present invention there is provideduse of a compound of formula (I), or a pharmaceutically acceptable saltthereof, in the manufacture of a medicament for the prophylaxis ortreatment of cancer.

According to a fifth aspect of the present invention, there is provideda method of prophylaxis or treatment of cancer comprising administrationto a patient in need of such treatment an effective dose of a compoundof formula (I), or a pharmaceutically acceptable salt thereof.

With respect to each of the third, fourth, and fifth aspects of thepresent invention, embodiments of the invention comprise a cancerselected from but not restricted to the group consisting of: pancreaticcancer, bladder cancer, other urothelial cancers (e.g. cancers of ureterand renal pelvis), gastrointestinal cancers (also known as cancer of thedigestive tract, including: oesophageal cancer, gastric cancer, stomachcancer, bowel cancer, small intestine cancer, colon cancer, colorectalcancer, appendix mucinous, goblet cell carcinoid, liver cancer, biliarycancer, gallbladder cancer, anal cancer and rectal cancer), lung cancer,renal (or kidney) cancer, biliary cancer, prostate cancer,cholangiocarcinoma, neuroendocrine cancer, sarcoma, lymphoma, thymiccancer, glioblastoma multiforme, a cancer of an unknown primary origin,mesothelioma, adrenal cancer, testicular cancer, cancer of the centralnervous system, basal cell carcinoma, Bowens disease, other skin cancers(such as malignant melanoma, merckel cell tumour and rare appendagetumours), ocular surface squamous neoplasia, germ cell tumours,leukaemia, multiple myeloma, lung cancer, liver cancer, breast cancer,head and neck cancer, neuroblastoma, thyroid carcinoma, oral squamouscell carcinoma, urinary bladder cancer, Leydig cell tumour, andgynaecological cancers (including ovarian cancer, cancer of thefallopian tube, peritoneal cancer, endometrial cancer, uterine cancerand cervical cancer, including epithelia cervix carcinoma).

In certain embodiments, the cancer is a leukaemia or a lymphoma.

The cancer may be a leukaemia.

There are four main types of leukaemia depending on whether they arechronic or acute, and myeloid or lymphoid in origin. These are: acutemyeloblastic leukaemia (AML), acute lymphoblastic leukaemia (ALL),chronic myelogenous leukaemia (CML) and chronic lymphocytic leukaemia(CLL).

Mixtures of these are also known; e.g. biphenotypic acute leukaemia(BAL; which is a mix of AML and ALL).

In particular embodiments, the leukaemia is selected from the groupconsisting of: acute myeloblastic leukaemia (AML), acute lymphoblasticleukaemia (ALL), chronic myelogenous leukaemia (CML), chroniclymphocytic leukaemia (CLL) and biphenotypic acute leukaemia (BAL; whichis a mix of AML and ALL).

Chronic lymphocytic leukaemia (CLL) includes the subtypes: B-cell CLL(B-CLL), B-cell prolymphocytic leukaemia (PLL) and T-cell chronicprolymphocytic leukaemia (T-PLL) and several subtypes that differ at thegenetic level.

Acute lymphoblastic leukaemia (ALL) includes the subtypes: precursorB-cell ALL (B ALL), precursor T-cell ALL (T-ALL), Burkitt-type ALL andPhiladelphia chromosome positive (BCR-ABL fusion) ALL.

Acute myeloblastic leukaemia (AML) includes the subtypes: myeloidleukaemia, monocytic leukaemia and acute promyelocytic leukaemia (APL).

Chronic myelogenous leukaemia (CML) includes the subtypes: chronicgranulocytic leukaemia (CGL) (CGL), Juvenile CML (JCML), chronicneutrophilic leukaemia (CNL), chronic myelomonocytic leukaemia (CMML)and atypical CML (aCML).

In particular embodiments, any of the above sub-types of CLL, ALL, AMLand CML are suitable for treatment with the compounds of the inventionor pharmaceutical compositions containing them

Other forms of leukemia that can be considered to sit outside of thesefour main groups include: erythroleukemia, arising from red blood cellprecursors; and a lymphoma that has gone into the blood.

The cancer may be a lymphoma, e.g. a solid lymphoma.

There are two main types of lymphoma: Hodgkin's lymphoma andnon-Hodgkin's lymphoma. Within each of these there are various subtypes.

In particular embodiments, the lymphoma is selected from the groupconsisting of: Hodgkin's lymphoma and non-Hodgkin's lymphoma.

In particular embodiments, the lymphoma is selected from the groupconsisting of: Burkitt's lymphoma (BL), mantle cell lymphoma (MCL),follicular lymphoma (FL), small lymphocytic lymphoma (SLL) indolentB-cell non-Hodgkin's lymphoma, histiocytic lymphoma (aka immunoblasticlymphoma; IBL) and diffuse large B-cell lymphoma (DLBCL), includingactivated-cell like diffuse large B-cell lymphoma (DLBCL-ABC) andgerminal center B-cell like diffuse large B-cell lymphoma (DLBCL-GCB).

In certain embodiments, the cancer is a gastrointestinal cancer and canbe selected from the group consisting of: oesophageal cancer, gastriccancer, stomach cancer, bowel cancer, small intestine cancer, coloncancer, colorectal cancer, appendix mucinous, goblet cell carcinoid,liver cancer, biliary cancer, gallbladder cancer, anal cancer and rectalcancer.

In certain embodiments, the cancer is of gynaecological origin and canbe selected from the group consisting of: a cancer of the uterus, cancerof the fallopian tube, cancer of the endometrium, cancer of the ovary,cancer of the peritoneum and cancer of the cervix). Suitably the ovariancancer may be epithelial ovarian cancer. Suitably the peritoneal cancermay be primary peritoneal cancer.

In particular, the cancer may be selected from, but not restricted topancreatic cancer, lung cancer, bladder cancer, breast cancer, biliarycancer, lymphoma, a leukaemia, a gastrointestinal cancer and agynaecological cancer.

The compounds of the invention have been found to retain activity evenunder hypoxic conditions. Cancers particularly associated with hypoxiaare pancreatic and renal (or kidney) cancers.

The cancer may be relapsed. The cancer may be metastatic. The cancer maybe previously untreated.

The cancer may be refractory cancer that has previously been treated buthas proven unresponsive to prior treatment. Alternatively, the cancerpatient may be intolerant of a previous therapy, for example, maydevelop side effects that make the patient intolerant to furthertreatment with the agent being administered.

According to a sixth aspect of the present invention, there is provideda pharmaceutical composition comprising a compound of formula (I), or apharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable excipient. The pharmaceutical compositionmay be for use in the prophylaxis or treatment of cancer, e.g. a canceror group of cancers mentioned above.

The compound of formula (I) may be as described in the followingnumbered paragraphs:

1. In a first aspect of the invention is provided a compound of formula(I), or a pharmaceutically acceptable salt thereof:

whereinR¹ is independently at each occurrence selected from: C₁-C₂₄-alkyl,C₃-C₂₄-alkenyl, C₃-C₂₄-alkynyl, C₀-C₄-alkylene-C₃-C₆-cycloalkyl orC₀-C₄-alkylene-aryl;R² and R³ are each independently at each occurrence selected from H,C₁-C₆-alkyl and C₁-C₃-alkylene-R⁷; or R² and R³ together with the atomto which they are attached form a 3- to 6-membered cycloalkyl orheterocycloalkyl group;R⁴ is independently at each occurrence H or C₁-C₄-alkyl;R⁵ is independently at each occurrence selected from aryl and 5-, 6-, 9-or 10-membered heteroaryl;R⁶ is independently selected from:

R⁷ is independently at each occurrence selected from aryl, imidazole,indole, SR^(a), ORE, CO₂R^(a), CO₂NR^(a)R^(a), NR^(a)R^(b) andNH(═NH)NH₂;R⁸ is independently selected from H and

Y is independently selected from H, F, Cl and OMe;X is independently at each occurrence a pharmaceutically acceptablecation;wherein any aryl group is either phenyl or naphthyl;wherein where any of R¹, R², R³, R⁴, R⁵ or R⁷ is an alkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl, that alkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl group is optionally substitutedwith from 1 to 4 substituents selected from: halo, nitro, cyano,NR^(a)R^(a), NR^(a)S(O)₂R^(a), NR^(a)C(O)R^(a), NR^(a)CONR^(a)R^(a),NR^(a)CO₂R^(a), OR^(a); SR^(a), SOR^(a), SO₃R^(a), SO₂R^(a),SO₂NR^(a)R^(a), CO₂R^(a), C(O)R^(a), CONR^(a)R^(a),CR^(a)R^(a)NR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl andC₁-C₄-haloalkyl; wherein R^(a) is independently at each occurrenceselected from: H and C₁-C₄-alkyl; and R^(b) is independently at eachoccurrence selected from: H, and C₁-C₄-alkyl and C(O)—C₁-C₄-alkyl.2. A compound of paragraph 1, wherein R⁴ is H.3. A compound of paragraph 1 or paragraph 2, wherein R⁶ is

4. A compound of paragraph 1 or paragraph 2, wherein R⁶ is

5. A compound of paragraph 1 or paragraph 2, wherein R⁶ is

6. A compound of paragraph 1 or paragraph 2, wherein R⁶ is

7. A compound of paragraph 6, wherein Y is H.8. A compound of paragraph 6, wherein Y is F.9. A compound of any one of paragraphs 6 to 8, wherein R⁸ is H.

10. A compound of any one of paragraphs 6 to 8, wherein R⁸ is

11. A compound of paragraph 1 or paragraph 2, wherein R⁶ is

12. A compound of paragraph 1 or paragraph 2, wherein R⁶ is

13. A compound of paragraph 1 or paragraph 2, wherein R⁶ is

14. A compound of any one of paragraphs 1 to 13, wherein R¹ is selectedfrom C₅-C₇-cycloalkyl, C₁-C₈-alkyl and benzyl.15. A compound of paragraph 14, wherein R¹ is benzyl.16. A compound of paragraph 14, wherein R¹ is C₁-C₈-alkyl, e.g., ethyl.17. A compound of any one of paragraphs 1 to 16, wherein R³ is H.18. A compound of any one of paragraphs 1 to 17, wherein R² isC₁-C₄-alkyl.19. A compound of any one of paragraphs 1 to 17, wherein R² is H.20. A compound of any one of paragraphs 1 to 19, wherein R⁵ is phenyl.21. A compound of any one of paragraphs 1 to 19, wherein R⁵ is naphthyl.22. A compound of any one of paragraphs 1 to 21 wherein r is a metalcation or an ammonium cation.23. A compound of any one of paragraphs 1 to 22 for medical use.24. A compound of any one of paragraphs 1 to 23 for use in treatingcancer.25. A compound for use of paragraph 24, wherein the cancer is leukaemiaor lymphoma.26. A compound for use of paragraph 25, wherein the cancer is aleukaemia selected from the group consisting of acute lymphoblasticleukaemia, acute myelogenous leukaemia, myelodysplastic syndromes, acutepromyelocytic leukaemia, acute lymphocytic leukaemia, chronicmyelogenous leukaemia, chronic lymphocytic leukaemia, monoblasticleukaemia and hairy cell leukaemia.27. A pharmaceutical composition comprising compounds of any one ofclaims 1 to 22 and at least one pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention are further described hereinafter withreference to the accompanying drawings, in which:

FIG. 1 shows sigmoidal dose-response curves for (A) clofarabine, (B)Example 1, and (C)

Example 2. Clofarabine and Example 1 showed the highest potency. Allassays were carried out using KGIa cells and data are presented as mean(±SD) of three independent experiments

FIG. 2 shows analysis of the LSC targeting capacity of clofarabine,Example 1, and Example 2. Under normoxic conditions, all of thecompounds showed stem cell targeting at concentrations above 1×10⁻⁷M.All data are the mean (±SD) of three independent experiments.

FIG. 3 shows comparison of the fraction of KG1a cells expressing an LSCphenotype under normoxic and hypoxic conditions. (A) KG1a cells show atime-dependent increase in cells expressing an LSC phenotype underhypoxic conditions, which was (B) reversed when the cells weretransferred into normoxic culture conditions. All data are the mean(±SD) of four independent experiments.

FIG. 4 shows the mean LD₅₀ values for (A) clofarabine, (B) Example 1,(C) Example 2, and (D) a comparison under normoxic and hypoxicconditions. Clofarabine showed a significant loss in potency underhypoxic conditions. In contrast, the compounds of the inventionmaintained their potency under hypoxic conditions. All data are the mean(±SD) of three independent experiments.

FIG. 5 shows comparison of the DNA damage, as measured by γH2A.Xphosphorylation assay, induced in purified LSCs and bulk tumour cellsfollowing 2-hour exposure to (A) clofarabine, (B) Example 1 and (C)Example 2 under hypoxic conditions (5% 02). All data are the mean (±SD)of three independent experiments.

DETAILED DESCRIPTION

The term ‘saline’ is intended to refer to an aqueous solution of sodiumchloride. Saline solutions of the present invention will typically besterile and will typically be at a concentration suitable for use inparenteral administration. Suitable concentrations are up to 2 w/v % orup to 1 w/v %. To optimise osmolarity different concentrations of salinecan be used in the formulations of the invention, e.g., 0.9% or 0.45%.

The formulations of the present invention can be used in the treatmentof the human body. They may be used in the treatment of the animal body.

The compounds in the formulations of the invention may be obtained,stored and/or administered in the form of a pharmaceutically acceptablesalt. Suitable pharmaceutically acceptable salts include, but are notlimited to, salts of pharmaceutically acceptable inorganic acids such ashydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic,and hydrobromic acids, or salts of pharmaceutically acceptable organicacids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic,benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic,stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic andvaleric acids. Suitable base salts are formed from bases which formnon-toxic salts. Examples include the aluminium, arginine, benzathine,calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium,meglumine, olamine, potassium, sodium, tromethamine and zinc salts.Hemisalts of acids and bases may also be formed, for example,hemisulfate, hemioxalate and hemicalcium salts.

For the above-mentioned formulations of the invention the dosageadministered will, of course, vary with the compound employed, theprecise mode of administration, the treatment desired and the disorderindicated. Dosage levels, dose frequency, and treatment durations ofcompounds of the invention are expected to differ depending on theformulation and clinical indication, age, and co-morbid medicalconditions of the patient. The size of the dose for therapeutic purposesof compounds of the invention will naturally vary according to thenature and severity of the conditions, the age and sex of the animal orpatient and the route of administration, according to well knownprinciples of medicine.

A pharmaceutical formulation typically takes the form of a compositionin which active compounds, or pharmaceutically acceptable salts thereof,are in association with a pharmaceutically acceptable adjuvant, diluentor carrier. One such pharmaceutically acceptable adjuvant, diluent orcarrier in the formulations of the invention is the polar aproticsolvent. Conventional procedures for the selection and preparation ofsuitable pharmaceutical formulations are described in, for example,“Pharmaceuticals —The Science of Dosage Form Designs”, M. E. Aulton,Churchill Livingstone, 1988.

The formulations may be suitable for topical application (e.g., to theskin or bladder), for oral administration or for parenteral (e.g.intravenous administration).

Any solvents used in pharmaceutical formulations of the invention shouldbe pharmaceutical grade, by which it is meant that they have an impurityprofile which renders them suitable for administration (e.g.,intravenous administration) to humans.

For oral administration the formulations of the invention may comprisethe active compound admixed with an adjuvant or a carrier, for example,lactose, saccharose, sorbitol, mannitol; a starch, for example, potatostarch, corn starch or amylopectin; a cellulose derivative; a binder,for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, forexample, magnesium stearate, calcium stearate, polyethylene glycol, awax, paraffin, and the like, and then compressed into tablets. If coatedtablets are required, the cores, prepared as described above, may becoated with a concentrated sugar solution which may contain, forexample, gum arabic, gelatine, talcum and titanium dioxide.Alternatively, the tablet may be coated with a suitable polymerdissolved in a readily volatile organic solvent.

For the preparation of soft gelatine capsules, the active compounds maybe admixed with, for example, a vegetable oil or polyethylene glycol.Hard gelatine capsules may contain granules of the compound using eitherthe above-mentioned excipients for tablets. Also liquid or semisolidformulations of the active compounds may be filled into hard gelatinecapsules.

Liquid preparations for oral application may be in the form of syrups orsuspensions, for example, solutions containing the compound of theinvention, the balance being sugar and a mixture of ethanol, water,glycerol and propylene glycol. Optionally such liquid preparations maycontain colouring agents, flavouring agents, sweetening agents (such assaccharine), preservative agents and/or carboxymethylcellulose as athickening agent or other excipients known to those skilled in art.

The formulations may be for parenteral (e.g., intravenous)administration. For parenteral (e.g., intravenous) administration theactive compounds may be administered as a sterile aqueous or oilysolution. Preferably, the active compounds are administered as a sterileaqueous solution. The aqueous solution may further comprise at least onesurfactant and/or organic solvent. Illustrative organic solvents includedimethylacetamide, ethanol, ethyleneglycol, N-methyl-pyrrolidinone,dimethylsulfoxide, dimethylformamide and isopropanol. Illustrativesurfactants include polyethoxylated fatty acids and fatty acid estersand mixtures thereof. Suitable surfactants include polyethoxylatedcastor oil (e.g., that sold under the trade name Kolliphor® ELP); orpolyethoxylated stearic acid (e.g. that sold under the trade namesSolutol® or Kolliphor® HS15); or polyethoxylated (e.g. polyoxyethylene(20)) sorbitan monooleate, (e.g. that are sold under the trade namesPolysorbate 80 or Tween® 80).

The pharmaceutical composition of the invention will preferably comprisefrom 0.05 to 99% w (percent by weight) compound of the invention, morepreferably from 0.05 to 80% w compound of the invention, still morepreferably from 0.10 to 70% w compound of the invention, and even morepreferably from 0.10 to 50% w compound of the invention, all percentagesby weight being based on total composition.

Cyclodextrins have been shown to find wide application in drug delivery(Rasheed et al, Sci. Pharm., 2008, 76, 567-598). Cyclodextrins are afamily of cyclic oligosaccharides. They act as a ‘molecular cage’ whichencapsulates drug molecules and alters properties of those drugmolecules such as solubility. Cyclodextrins comprise (α-1,4)-linkedα-D-glucopyranose units. Cyclodextrins may contains 6, 7 or 8glucopyranose units (designated α-, β- and γ-cyclodextrinsrespectively). Cyclodextrins used in pharmaceutical formulations areoften β-cyclodextrins. The pendant hydroxyl groups can be alkylated witha C₁-C₆ substituted or unsubstituted alkyl group. Examples ofcyclodextrins are α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,2-hydroxypropyl-β-cyclodextrin (HP-β-CD), sulfobutylether β-cyclodextrinsodium salt, partially methylated β-cyclodextrin. The formulations ofthe invention may also comprise at least one cyclodextrin.

The term C_(m)-C_(n) refers to a group with m to n carbon atoms.

The term “alkyl” refers to a linear or branched hydrocarbon group. Analkyl group is monovalent. For example, C₁-C₆-alkyl may refer to methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyland n-hexyl. The alkyl groups are preferably unsubstituted.

The term “alkylene” refers to a linear hydrocarbon chain. An alkylenegroup is divalent. For example, C₁-alkylene may refer to a CH₂ group.C₂-alkylene may refer to —CH₂CH₂-group. The alkylene groups arepreferably unsubstituted.

The term “haloalkyl” refers to a hydrocarbon chain substituted with atleast one halogen atom independently chosen at each occurrence from:fluorine, chlorine, bromine and iodine. The halogen atom may be presentat any position on the hydrocarbon chain. For example, C₁-C₄-haloalkylmay refer to chloromethyl, fluoromethyl, trifluoromethyl, chloroethyle.g., 1-chloromethyl and 2-chloroethyl, trichloroethyl e.g.,1,2,2-trichloroethyl, 2,2,2-trichloroethyl, fluoroethyl e.g.,1-fluoromethyl and 2-fluoroethyl, trifluoroethyl e.g.1,2,2-trifluoroethyl and 2,2,2-trifluoroethyl, chloropropyl,trichloropropyl, fluoropropyl, trifluoropropyl. A halo alkyl group maybe a fluoroalkyl group, i.e., a hydrocarbon chain substituted with atleast one fluorine atom.

The term “alkenyl” refers to a branched or linear hydrocarbon chaincontaining at least one carbon-carbon double bond. The double bond(s)may be present as the E or Z isomer. The double bond may be at anypossible position of the hydrocarbon chain. For example, “C₂-C₄-alkenyl”may refer to ethenyl, ally and butenyl. The alkenyl groups arepreferably unsubstituted.

The term “alkynyl” refers to a branched or linear hydrocarbon chaincontaining at least one carbon-carbon triple bond. The triple bond maybe at any possible position of the hydrocarbon chain. For example,“C₂-C₆-alkynyl” may refer to ethynyl, propynyl, butynyl. The alkynylgroups are preferably unsubstituted.

The term “cycloalkyl” refers to a saturated hydrocarbon ring systemcontaining 3, 4, 5 or 6 carbon atoms. For example, “3- to 6-memberedcycloalkyl” may refer to cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl. The cycloalkyl groups are preferably unsubstituted.

The term “heterocycloalkyl” may refer to a saturated or partiallysaturated monocyclic group comprising 1 or 2 heteroatoms independentlyselected from O, S and N in the ring system (in other words 1 or 2 ofthe atoms forming the ring system are selected from O, S and N).Examples of heterocycloalkyl groups include; piperidine, piperazine,morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran,tetrahydrothiophene, tetrahydropyran, dihydropyran, dioxane, azepine.The heterocycloalkyl groups are preferably unsubstituted or substituted.

The present invention also includes formulations of all pharmaceuticallyacceptable isotopically-labelled forms of compound wherein one or moreatoms are replaced by atoms having the same atomic number, but an atomicmass or mass number different from the atomic mass or mass number of thepredominant isotope usually found in nature.

Examples of isotopes suitable for inclusion in the compounds of theinvention include isotopes of hydrogen, such as ²H and ³H, carbon, suchas ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F,iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, oxygen,such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulphur, such as³⁵S.

Certain isotopically-labelled compounds, for example, thoseincorporating a radioactive isotope, are useful in drug and/or substratetissue distribution studies. The radioactive isotopes tritium, i.e. ³H,and carbon-14, i.e. ¹⁴C, and ¹⁸F are particularly useful for thispurpose in view of their ease of incorporation and ready means ofdetection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Isotopically-labelled compounds can generally be prepared byconventional techniques known to those skilled in the art or byprocesses analogous to those described using an appropriateisotopically-labelled reagent in place of the non-labelled reagentpreviously employed.

The method of treatment or the formulation for use in the treatment ofcancer, including lymphoma or leukaemia, may involve, in addition to theformulations of the invention, conventional surgery or radiotherapy orchemotherapy. Such chemotherapy may include the administration of one ormore other active agents.

Where a further active agent is administered as part of a method oftreatment of the invention, such combination treatment may be achievedby way of the simultaneous, sequential or separate dosing of theindividual components of the treatment. Such combination products employthe compounds of this invention within a therapeutically effectivedosage range described hereinbefore and the one or more otherpharmaceutically-active agent(s) within its approved dosage range.

Thus, the pharmaceutical formulations of the invention may compriseanother active agent.

The one or more other active agents may be one or more of the followingcategories of anti-tumour agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof,such as alkylating agents (for example cyclophosphamide, nitrogenmustard, bendamustin, melphalan, chlorambucil, busulphan, temozolamideand nitrosoureas); antimetabolites (for example gemcitabine andantifolates such as fluoropyrimidines like 5-fluorouracil and tegafur,raltitrexed, methotrexate, pemetrexed, cytosine arabinoside, andhydroxyurea); antibiotics (for example anthracyclines like adriamycin,bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere and polokinase inhibitors); proteasomeinhibitors, for example carfilzomib and bortezomib; interferon therapy;and topoisomerase inhibitors (for example epipodophyllotoxins likeetoposide and teniposide, amsacrine, topotecan, mitoxantrone andcamptothecin);(ii) cytostatic agents such as antiestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5α-reductase suchas finasteride;(iii) anti-invasion agents, for example dasatinib and bosutinib(SKI-606), and metalloproteinase inhibitors, inhibitors of urokinaseplasminogen activator receptor function or antibodies to Heparanase;(iv) inhibitors of growth factor function: for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies,for example the anti-erbB2 antibody trastuzumab [Herceptin™], theanti-EGFR antibody panitumumab, the anti-erbB 1 antibody cetuximab,tyrosine kinase inhibitors, for example inhibitors of the epidermalgrowth factor family (for example EGFR family tyrosine kinase inhibitorssuch as gefitinib, erlotinib and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine(CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib);inhibitors of the hepatocyte growth factor family; inhibitors of theinsulin growth factor family; modulators of protein regulators of cellapoptosis (for example Bcl-2 inhibitors); inhibitors of theplatelet-derived growth factor family such as imatinib and/or nilotinib(AMN107); inhibitors of serine/threonine kinases (for example Ras/Rafsignalling inhibitors such as farnesyl transferase inhibitors, forexample sorafenib, tipifarnib and lonafarnib), inhibitors of cellsignalling through MEK and/or AKT kinases, c-kit inhibitors, abl kinaseinhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinaseinhibitors, IGF receptor, kinase inhibitors; aurora kinase inhibitorsand cyclin dependent kinase inhibitors such as CDK2 and/or CDK4inhibitors;(v) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, [for example the anti-vascularendothelial cell growth factor antibody bevacizumab (Avastin™);thalidomide; lenalidomide; and for example, a VEGF receptor tyrosinekinase inhibitor such as vandetanib, vatalanib, sunitinib, axitinib andpazopanib;(vi) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2;(vii) immunotherapy approaches, including for example adoptive celltransfer, such as CAR T-cell therapy; antibody therapy such asalemtuzumab, rituximab, ibritumomab tiuxetan (Zevalin®) and ofatumumab;interferons such as interferon α; interleukins such as 1L-2(aldesleukin); interleukin inhibitors for example IRAK4 inhibitors;cancer vaccines including prophylactic and treatment vaccines such asHPV vaccines, for example Gardasil, Cervarix, Oncophage and Sipuleucel-T(Provenge); and toll-like receptor modulators for example TLR-7 or TLR-9agonists;(viii) cytotoxic agents for example fludarabine (fludara), cladribine,pentostatin (Nipent™);(ix) steroids such as corticosteroids, including glucocorticoids andmineralocorticoids, for example aclometasone, aclometasone dipropionate,aldosterone, amcinonide, beclomethasone, beclomethasone dipropionate,betamethasone, betamethasone dipropionate, betamethasone sodiumphosphate, betamethasone valerate, budesonide, clobetasone, clobetasonebutyrate, clobetasol propionate, cloprednol, cortisone, cortisoneacetate, cortivazol, deoxycortone, desonide, desoximetasone,dexamethasone, dexamethasone sodium phosphate, dexamethasoneisonicotinate, difluorocortolone, fluclorolone, flumethasone,flunisolide, fluocinolone, fluocinolone acetonide, fluocinonide,fluocortin butyl, fluorocortisone, fluorocortolone, fluocortolonecaproate, fluocortolone pivalate, fluorometholone, fluprednidene,fluprednidene acetate, flurandrenolone, fluticasone, fluticasonepropionate, halcinonide, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone valerate, icomethasone, icomethasone enbutate,meprednisone, methylprednisolone, mometasone paramethasone, mometasonefuroate monohydrate, prednicarbate, prednisolone, prednisone,tixocortol, tixocortol pivalate, triamcinolone, triamcinolone acetonide,triamcinolone alcohol and their respective pharmaceutically acceptablederivatives. A combination of steroids may be used, for example acombination of two or more steroids mentioned in this paragraph;(x) targeted therapies, for example PI3Kd inhibitors, for exampleidelalisib and perifosine; or checkpoint inhibitor compounds includinganti-PD-1, anti-PD-L1 and anti-CTLA4 molecules, such as nivolumab,pembrolizumab, pidilizumab, atezolizumab, durvalumab and avelumab.

The one or more other active agents may also be antibiotic.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or process so disclosed, may be combined inany combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive. The invention is notrestricted to the details of any foregoing embodiments. The inventionextends to any novel one, or any novel combination, of the featuresdisclosed in this specification (including any accompanying claims,abstract and drawings), or to any novel one, or any novel combination,of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which arefiled concurrently with or previous to this specification in connectionwith this application and which are open to public inspection with thisspecification, and the contents of all such papers and documents areincorporated herein by reference.

Examples

Throughout the specification, these abbreviations have the followingmeanings.

AcCN acetonitrile aq aqueous DCM dichloromethane DMSO dimethylsulphoxideEC₅₀ Half maximal effective concentration eq. equivalents HPLC highpressure liquid chromatography h hours IC₅₀ half maximal inhibitoryconcentration MS mass spectrometry NMR nuclear magnetic resonance Np1-naphthyl rt room temperature TEA triethylamine TEAB triethylammoniumhydrogen carbonate buffer TEP triethyl phosphate t_(R) retention timeGeneral procedure for the synthesis of diphosphate phosphoramidates

Dry nucleoside A (˜1.0 mmol, 1.0 eq.) was dissolved in 5 mL oftriethylphosphate (TEP) and stirred for 5 min at room temperature. Thereaction mixture was cooled to 4° C. and dry1,8-bis(dimethylamino)naphthalene (Proton Sponge®, 1.5 eq.) was added,followed by dropwise addition of POC13 (1.3 eq.). The resultingsuspension was stirred at 4° C. for 2-3 hours. After this time, to thereaction mixture was added cold 0.5 M aq. triethylammonium hydrogencarbonate buffer (TEAB) (pH 7.5) and the resulting mixture was stirredfor 30 minutes and then allowed to warm to room temperature and stirredfor additional 1 h. TEP was extracted with tert-butylmethyl ether (4×50mL), and aqueous solution was evaporated and dried under high vacuumovernight to yield the corresponding nucleoside 5′-monophosphatetriethylammonium salt B as glassy colourless oil or white powder, whichwas used in the next step without further purification. The formation ofthe nucleoside 5′-monophosphate was confirmed by negative ion massspectrometry.

A 0.5 M solution of TEAB was prepared by bubbling CO₂ through a 0.5 Mtriethylamine (TEA) solution in water at 0-4° C. for 30-45 min (pH ofapproximately 7.4-7.6, unless stated otherwise)

A synthesis of nucleoside 5′-monophosphate triethylammonium salt in thefirst step was accomplished following modified reference: El-Tayeb A. etal. J. Med. Chem. 2006, 49(24), 7076 7087.

A mixture of nucleoside 5′-monophosphate triethylammonium salt B (1mmol) in 1:1 mixture of 1,4-dioxane/acetonitrile (20 mL) was stirred atroom temperature (rt) under an argon atmosphere and an appropriatephosphorochloridate C (1.0 eq.) dissolved in anhydrous acetonitrile wasadded dropwise. The resulting reaction mixture was stirred for 18 h atrt. The crude mixture was evaporated under reduced pressure and theresidue was re-dissolved in DCM and adsorbed on silica gel deactivatedwith TEA (3%) in a mixture of DCM/MeOH (92%/5%). Purification of crudematerials (prepared for dry load) by automatic Biotage Isolera One onSNAP Ultra 50 g cartridge pre-primed with 3CV of TEA (3%) in a mixtureof DCM/MeOH (92%/5%) with a gradient of McOH in DCM (2% to 20%, flow 50mL/min) afforded desired compounds, which were further re-purified onBiotage Isolera One (C₁₈ SNAP Ultra 30 g cartridge with gradient of 0.1M TEAB in AcCN (acetonitrile) from 100 to 0% as an eluent in 60 min.,flow 15 mL/min.) or by HPLC on semi-preparative column (Varian PursuitXRs 5C18, 150×21.22 mm) using gradient of 0.1 M TEAB in AcCN from 90/10to 0/100 in 30 min.

Example 1:2-Chloro-9-(2′-Deoxy-2′-fluoro-11-D-arabinofuranosyl)adenine-5′-O-[phenyl(benzoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.58 g, 0.98 mmol),phenyl(benzoxy-L-alaninyl)phosphorochloridate (0.35 g, 0.98 mmol) as awhite foam (0.04g, 6%).

³¹P NMR (202 MHz, MeOD): δ_(P) −7.73 (d, J=21.0 Hz), −7.93 (d, J=21.2Hz),−11.96 (d, J=18.4 Hz),−12.06 (d, J=20.8 Hz). ¹H NMR (500 MHz, MeOD):δ_(H) 8.29,8.28 (2×0.5H, H-8), 7.33-7.24 (9H, m, H-Ar), 7.18-7.14 (1H,m, H-Ar), 6.44,6.41 (2×0.5H, 2×d, J=3.5 Hz, H-1′), 5.20-5.19 (0.5H, m,H-2′), 5.11 (2H, apparent s, OCH₂Ph), 5.10-5.08 (0.5H, m, H-2′),4.58-4.54 (1H, m, H-3′), 4.26-4.24 (2H, m, 2×H-5′), 4.16-4.13 (2H, m,H-4′, NHCHCH₃), 3.08 (6H, q, J=7.0 Hz, 3×CH₂CH₃), 1.39, 1.35 (3H, 2×d,J=7.0 Hz, NHCHCH₃), 1.26 (9H, t, J=7.0 Hz, 3×CH₂CH₃). MS (ES) negativemode m/z: 699 ([M-H]⁻), Accurate mass: C₂₅H₂₈ClFN₆O₁₀P₂ required 700.93found 699.20 ([M-H]⁻) Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCNfrom 90/10 to 0/100 in 30 min, F=1 mL/min, λ=254 nm, t_(R) =14.05 min.

Example 2:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[phenyl(ethoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.58 g, 0.98 mmol), phenyl(ethoxy-L-alaninyl)phosphorochloridate (0.28 g, 0.98 mmol) as a whitesolid (0.07 g, 10%).

³¹P NMR (202 MHz, MeOD): δ_(P) −7.72 (d, J=20.2 Hz),−7.89 (d, J=20.2Hz),−11.94 (d, J=18.2 Hz),−12.10 (d, J=18.2 Hz). ¹H NMR (500 MHz, MeOD):δ_(H) 8.29 (1H, s, H-8), 7.34-7.27 (4H, m, H-Ar), 7.17-7.15 (1H, m,H-Ar), 6.45, 6.42 (2×0.5H, 2 ×apparent t, J=1.5 Hz, H-1′), 5.24, 5.10(2×0.5H, 2 ×apparent t, J=1.5 Hz, H-2′), 4.59, 4.55 (2×0.5H, 2 ×apparentt, J=2.0 Hz, H-3′), 4.26-4.24 (2H, m, OCH₂CH₃), 4.17-4.07 (4H, m, H-5′,NHCHCH₃, H-4′), 3.05 (6H, q, J=7.0 Hz, 3×CH₂CH₃), 1.39, 1.34 (2×1.5H,2×d, J=7.0 NHCHCH₃), 1.26-1.20 (12H, m, OCH₂CH₃, 3 ×CH₂CH₃). MS (ES)negative mode m/z: 637 ([M-H]⁻), Accurate mass: C₂₁H₂₆ClFN₆O₁₀P₂required 638.86 found 637.12 ([M-H]⁻) Reverse-phase HPLC, eluting withH₂O/AcCN from 90/10 to 0/100 in 30 min, F=1 mL/min, λ=254 nm, two peaksfor two diastereoisomers with t_(R) =14.20 min.

Example 3:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[phenyl(benzoxy-L-glycinyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.58 g, 0.98 mmol), phenyl(benzoxy-L-glycinyl)phosphorochloridate (0.335 g, 0.98 mmol) as a whitesolid (0.10 g, 13%).

³¹P NMR (202 MHz, MeOD): δ_(P) −6.70 (d, J=20.2 Hz), −11.86 (d, J=22.2Hz). ¹H NMR (500 MHz, MeOD): δ_(H) 8.30, 8.29 (2×0.5H, 2×d, J=2.0 Hz,H-8), 7.36-7.28 (9H, m, H-Ar), 7.18-7.14 (1H, m, H-Ar), 6.44, 6.41(2×0.5H, 2×d, J=4.5 Hz, H-1′), 5.20-5.18 (0.5H, m, H-2′), 5.16 (2H,apparent s, OCH₂Ph), 5.09-5.07 (0.5H, m, H-2′), 4.61-4.55 (1H, m, H-3′),4.29-4.25 (2H, m, 2×H-5′), 4.17-4.13 (1H, m, H-4′), 3.96-3.84 (2H, m,NHCH2), 2.89 (6H, q, J=7.0 Hz, 3×CH₂CH₃), 1.19 (9H, t, J=7.0 Hz,3×CH₂CH₃). MS (ES) negative mode m/z: 685 ([M-H]⁻), Accurate mass:C₂₅H₂₆ClFN₆O₁₀P₂ required 686 found 685.119 ([M-H]⁻). Reverse-phaseHPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in 30 min, F=1mL/min, λ=254 nm, t_(R)=13.6 min.

Example 4:5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(benzoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from5-fluoro-2′-deoxyuridine-5′-monophosphate TEA salt (1.07 g, 2.03 mmol),1-naphthyl(benzoxy-L-alaninyl)phosphorochloridate (0.82 g, 2.03 mmol) asa white solid (0.16 g, 10%).

³¹P NMR (202 MHz, MeOD): δ_(P) −7.43 (d, J=22.2 Hz), −7.79 (d, J=22.2Hz), −11.89 (d, J=20.2 Hz), −12.02 (d, J=20.2 Hz). ¹H-NMR (MeOD, 500MHz) δ_(H) 8.39-8.35 (1H, m, H-Ar), 7.99 (1H, apparent t, J=6.0 Hz,H-Ar), 7.87-7.86 (1H, m, H-Ar), 7.69 (1H, t, J=7.0 Hz, H-Ar), 7.58 (1H,apparent d, J=7.0 Hz, H-Ar), 7.53-7.51 (2H, m, H—Ar, H-6), 7.43-7.36(1H, m, H-Ar), 7.29-7.26 (5H, m, H-Ar), 6.26 (1H, apparent q, J=6.5 Hz,H-1′), 5.02-4.94 (2H, 2 ×apparent d, J=13.5, J=12.5 Hz, OCH₂Ph),4.46-4.43 (1H, m, H-3′), 4.24-4.16 (3H, m, 3H, 2 ×H-5′, NHCHCH₃), 4.03(1H, apparent s, H-4′), 3.09 (6H, q, J=7.0 Hz, 3×CH₂CH₃), 2.22-2.19 (1H,m, H-2′), 2.16-2.4 (1H, m, H-2′), 1.37 (1.5H, d, J=7.0 Hz, NHCHCH₃),1.29 (1.5H, d, J=7.0 Hz, NHCHCH₃), 1.26 (9H, t, J=7.0 Hz, 3×CH₂CH₃). MS(ES) negative mode m/z: 692 ([M-H]⁻), Accurate mass: C₂₉H₃₀FN₃O₁₂P₂required 693 found 692.128 ([M-H]⁻). Reverse-phase HPLC, eluting with0.1 M TEAB/AcCN from 90/10 to 0/100 in 30 min, F=1 mL/min, λ=254 nm,t_(R)=14.24 min., 14.34 min.

Example 5: 8-Chloroadenosine-5′-O-[1-naphthyl-(benzoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from8-chloroadenosine-5′-monophosphate TEA salt (0.46 g, 0.795 mmol),1-naphthyl(benzoxy-L-alaninyl)phosphorochloridate (0.32 g, 0.795 mmol)after two re-purifications on Biotage Isolera One (C₁₈ ₃₀ g cartridge)and HPLC (semi-preparative C₁₈ column) as a white solid (0.02 g, 4%).

³¹P NMR (202 MHz, MeOD): δ_(P) −7.58 (d, J=22.2 Hz), −8.0 (d, J=20.2Hz), −11.85 (d, J=22.2 Hz), −12.05 (d, J=20.2 Hz). ¹H NMR (500 MHz,CD₃OD): δ_(H) 8.36-8.33 (1H, m, H-Ar), 8.14, 8.10 (2×0.5H, 2 ×s, H-2),7.86-7.84 (1H, m, H-Ar), 7.66 (1H, d, J=8.0 Hz, H-Ar), 7.56-7.45 (3H, m,H-Ar), 7.38-7.33 (1H, m, H-Ar), 7.29-7.24 (5H, m, H-Ar), 6.02 (1H, t,J=6.0 Hz, H-1′), 5.35-5.32 (1H, m, H-2′), 5.00, 4.98, 4.97, 4.94 (2H,AB, JAB=12.3 Hz, OCH₂Ph), 4.54-4.50 (1H, m, H-3′), 4.40-4.33 (1H, m,H-5′), 4.26-4.13 (3H, m, H-5′, H-4′, NHCHCH₃), 3.06 (6H, q, J=7.0 Hz,3×CH₂CH₃), 1.36 (1.5H, d, J=7.0 Hz, NHCHCH₃), 1.26 (1.5H, d, J=7.0 Hz,NHCHCH₃), 1.25 (9H, t, J=7.0 Hz, 3×CH₂CH₃). MS (ES) negative mode m/z:747 ([M-H]⁻), Accurate mass: C₃₀H₃₁ClN₆O₁₁P₂ required 749 found 747.1102([M-H]⁻). Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to0/100 in 30 min, F=1 mL/min, λ=254 nm, t_(R)=14.32, 14.48 min.

Examples 6 and 7:3′-Deoxyadenosine-5′-O-[phenyl(benzoxy-L-alaninyl)]diphosphatetriethylammonium salt (6) and3′-Deoxyadenosine-5′,2′-bis-O-[phenyl(benzoxy-L-alaninyl)]-diphosphatetriethylammonium salt (7)

Prepared according to the general procedure from3′-deoxyadenosine-5′-monophosphate TEA salt (0.63 g, 1.186 mmol),phenyl(benzoxy-L-alaninyl)phosphorochloridate (0.42 g, 1.186 mmol).

Two products were isolated:3′-deoxyadenosine-5′,2′-O-[phenyl(benzoxy-L-alaninyl)]diphosphatetriethylammonium salt (0.05 g, 5%) and 3′-deoxyadenosine-5′[phenyl(benzoxy-L-alaninyl)] diphosphate as a colorless film (0.01 g,1.3%).

³¹P NMR (202 MHz, MeOD): δ_(P) −7.76 (d, J=21.0 Hz), −8.03 (d, J=21.8Hz), −11.70 (d, J=20.4 Hz), −11.81 (d, J=16.76 Hz). ¹H NMR (500 MHz,CD₃OD): δ_(H) 8.45 (1H, s, H-8), 8.21 (1H, s, H-2), 7.34-7.23 (9H, m,H-Ar), 7.14-7.11 (1H, m, H-Ar), 6.02 (1H, apparent s, H-1′), 5.10 (2H,AB system apparent t, J_(AB)=16.5 Hz, OCH₂Ph), 4.66-4.60 (2H, m, H-2′,H-4′), 4.29-4.27 (1H, m, H-5′), 4.19-4.13 (2H, m, H-5′, NHCHCH₃), 3.06(6H, q, J=7.0 Hz, 3×CH₂CH₃), 2.42-2.33 (1H, m, H-3′), 2.11— 2.06 (1H, m,H-3′), 1.40 (1.5H, d, J=7.0 Hz, NHCHCH₃), 1.35 (1.5H, d, J=7.0 Hz,NHCHCH₃), 1.25 (9H, t, J=7.0 Hz, 3×CH₂CH₃). MS (ES) negative mode m/z:647 ([M-H]⁻), Accurate mass: C₂₆H₃₀N₆O₁₀P₂ required 648 found 647.154([M-H]⁻). Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to0/100 in 20 min, F=1 mL/min, λ=254 nm, t_(R)=8.20 min.

³¹P NMR (202 MHz, MeOD): δ_(P) −7.78 (d, J=20.4 Hz), −7.84 (d, J=22.08Hz), −8.01 (d, J=21.0 Hz), −12.08 (d, J=21.6 Hz), −12.18 (d, J=22.0 Hz),−13.50 (d, J=20.6 Hz), −13.56 (d, J=20.2 Hz). ¹H NMR (500 MHz, CD₃OD):δ_(H) H 8.20 (0.5H, s, H-8), 8.19 (0.5H, s, H-8), 8.04 (1H, s, H-2),7.21-7.19 (12H, m, H-Ar), 7.14-6.97 (8H, m, H-Ar), 6.16 (0.5H, s, H-1′),6.15 (0.5H, s, H-1′), 5.27 (0.5H, apparent t, J=7.0 Hz, H-2′), 5.15(0.5H, apparent t, J=5.0 Hz, H-2′), 5.0-4.94 (4H, m, 2×OCH₂Ph),4.48-4.60 (1H, m, H-4′), 4.13-4.08 (1H, m, H-5′), 4.05-3.95 (3H, m,H-5′, 2×NHCHCH₃), 3.06 (12H, q, J=7.0 Hz, 3×CH₂CH₃), 2.50-2.42 (1H, m,H-3′), 2.27-2.20 (1H, m, H-3′), 1.26-1.17 (6H, m, 2×NHCHCH₃), 1.25 (18H,t, J=7.0 Hz, 3×CH₂CH₃). MS (ES) negative mode m/z: 1044 ([M-H]⁻),Accurate mass: C₄₂H₄₇N₇O₁₇P₄ required 1045 found 1044.232 ([M-H]⁻).Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, t_(R) =9.75 min, 10.37 min.

Example 8: 2′-Deoxy-2 ‘,2’-difluoro-D-cytidine-5′-O-[phenyl(benzoxy-L-alaninyl)] diphosphate triethylammonium salt

Prepared according to the general procedure from2′-deoxy-2′,2′-difluoro-D-cytidine 5′-monophosphate TEA salt (0.62 g,1.13 mmol), phenyl(benzoxy-L-alaninyl)phosphorochloridate (0.40 g, 1.13mmol) as a white solid (0.05 g, 6%).

³¹P NMR (202 MHz, MeOD): δ_(P)−7.65 (d, J=21.0 Hz), −7.87 (d, J=21.0Hz), −12.12 (d, J=16.9 Hz), −12.21 (d, J=21.4 Hz). ¹H NMR (500 MHz,MeOD): ox 7.83 (1H, d, J=7.5 Hz, H-6), 7.35-7.25 (9H, m, H-Ar), 7.16(1H, apparent t, J=7.0 Hz, H-Ar), 6.27-6.24 (1H, m, H-1′), 5.96, 5.95(1H, 2×d, J=5.0 Hz, H-5), 5.13 (2H, AB system, apparent s, OCH₂Ph),4.37-4.25 (3H, m, 2 ×H-5′, H-3′), 4.18-4.12 (1H, m, H-4′), 4.00-3.97(1H, m, NHCHCH₃), 3.20 (6H, q, J=7.0 Hz, 3×CH₂CH₃), 1.40, 1.35 (3H, 2×d,J=7.5 Hz, NHCHCH₃), 1.31 (9H, t, J=7.5 Hz, 3 ×CH₂CH₃). MS (ES) negativemode m/z: 659 ([M-H]⁻), Accurate mass: C₂₅H₂₈F₂N₄O₁₁P₄ required 660found 659 ([M-H]⁻). Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCNfrom 90/10 to 0/100 in 30 min, F=1 mL/min, λ=254 nm, t_(R) =12.55 min,12.85 min.

Example 9:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′[phenyl(neopentoxy-L-alaninyl)] diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-(3-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.77 g, 1.32 mmol), phenyl(neopentoxy-L-alaninyl)phosphorochloridate (0.43 g, 1.32 mmol) and TEA(0.53 mL, 3.81 mmol) as a white solid (0.065 g, 6%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)−7.67 (d, J=21.0 Hz), −7.93 (d, J=21.0Hz), −11.93 (d, J=21.21 Hz), −12.04 (d, J=21.21 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) H8.18 (0.5H, s, H-8), 8.17 (0.5H, s,H-8), 7.22-7.15 (4H, m, H-Ar), 7.12-7.09 (1H, m, H-Ar), 6.33 (0.5H, t,J=1.5 Hz, H-1′), 6.30 (0.5H, t, J=1.5 Hz, H-1′), 5.08 (0.5H, apparent t,J=1.5 Hz, H-2′), 4.96 (0.5H, apparent t, J=1.5 Hz, H-2′), 4.46 (0.5H,apparent t, J=2.0 Hz, H-3′), 4.43 (0.5H, apparent t, J=2.0 Hz, H-3′),4.15-4.11 (2H, m, H-5′), 4.05-3.99 (2H, m, NHCHCH₃, H-4′), 3.72, 3.69,3.63, 3.61 (2H, AB system, J=10.50 Hz, OCH₂C(CH₃)₃), 3.05 (6H, q, J=7.0Hz, N(CH₂CH₃)₃), 1.31, 1.25 (2×1.5H, 2×d, J=7.0 NHCHCH₃), 1.31 (9H, t,J=7.5 Hz, N(CH₂CH₃)₃), 0.80 (9H, s, OCH₂C(CH₃)₃).

¹³C NMR (125 MHz, CD₃OD): δ_(C) 175.10 (d,³J_(C-P)=6.4 Hz, C=0, ester),173.56 (d,³J_(C-P)=6.4 Hz, C═O, ester), 156.72 (C-6), 154.16 (C-2),153.42 (d, ²J_(C-P)=3.8 Hz, C—Ar), 150.90 (d, ²J_(C-P)=5.8 Hz, C-Ar),150.26 (C-4), 142.68 (C-8), 129.26, 128.73, 124.65, 124.60, 122.40(CH—Ar), 120.38, 120.15 (2×d, ³J_(C-P)=4.6 Hz, CH—Ar), 117.15 (C-5),94.99 (d, ¹J_(C-F)=191.4 Hz, C-2′), 82.92 (d, ³J_(C-F)=8.25 Hz, C-4′),82.78 (d, ²J_(C-F)=8.5 Hz, C-1′), 73.96, 73.66 (OCH₂C(CH₃)₃), 73.61 (d,²J_(C-F) =24.5 Hz, C-3′), 65.05 (C-5′), 50.59, 50.29 (NHCHCH₃), 46.26(N(CH₂CH₃)₃), 30.91 (OCH₂C(CH₃)₃), 25.36 (OCH₂C(CH₃)₃), 19.75, 19.63(2×d, ³J_(C-P)=5.4 Hz, NHCHCH₃), 7.84 (N(CH₂CH₃)₃).

C₂₄H₃₂ClFN₆O₁₀P₂ mass required m/z 680.94; found MS (ES⁻) m/z: 679.19([M-H]⁻)

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=15.83 min.

Example 10:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[phenyl(benzoxy-dimethylglycinyl)] diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.96 g, 1.64 mmol), phenyl(benzoxy-dimethylglycinyl)phosphorochloridate (0.60 g, 1.64 mmol) andTEA (0.66 mL, 4.75 mmol) as a white solid (0.085 g, 6%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-9.79 (d, J=22.6 Hz),−9.82 (d, J=22.6Hz),−12.17 (d, J=21.20 Hz),−12.20 (d, J=21.0 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.29 (0.5H, s, H-8), 8.27 (0.5H, s, H-8),7.36-7.26 (9H, m, H-Ar), 7.16-7.13 (1H, m, H-Ar), 6.43 (0.5H, d, J=1.5Hz, H-1′), 6.39 (0.5H, d, J=1.5 Hz, H-1′), 5.20-5.17 (0.5H, m, H-2′),5.15-5.13 (2H, m, OCH₂Ph), 5.11-5.07 (0.5H, m, H-2′), 4.60-4.52 (1H, m,H-3′), 4.26-4.23 (2H, m, H-5′), 4.15-4.11 (1H, m, H-4′), 3.05 (6H, q,J=7.0 Hz, N(CH₂CH₃)₃), 1.54 (6H, s, NHC(CH₃)₂), 1.31 (9H, t, J=7.5 Hz,N(CH₂CH₃)₃).

¹³C NMR (125 MHz, CD₃OD): δ_(C) 174.96 (d, ³J_(C-P)=6.6 Hz, C=0, ester),156.69 (C-6), 154.13 (C-2), 151.06 (d, ²J_(C-P)=7.3 Hz, C-Ar), 150.22(C-4), 140.21 (C-8), 136.04 (C-Ar), 129.15. 128.12. 127.76, 127.68,124.48, 120.47, 120.43 (CH—Ar), 117.12 (C-5), 95.49 (d, J_(C-F)=192.5Hz, C-2′), 82.84 (d, ³J_(C-F)=3.6 Hz, C-4′), 82.68 (d, ²J_(C-F)=11.5 Hz,C-1′), 73.59 (d, ²J_(C-F)=11.5 Hz, C-3′), 73.41 (d, ²J_(C-F)=10.5 Hz,C-3′), 66.74 (OCH₂Ph), 64.90 (C-5′), 48.47 (NHC(CH₃)₂), 46.35(N(CH₂CH₃)₃), 26.14, 26.12 (NHC(CH₃)2, 9.85 (N(CH₂CH₃)₃).

C₂₇H₃₀C₁FN₆O₁₀P₂ mass required m/z 714.96; found MS (ES⁻) m/z: 713.13([M—H]⁻)

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=14.51 min.

Example 11:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-naphthyl(benzoxy-dimethylglycinyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.96 g, 1.64 mmol),1-naphthyl(benzoxy-dimethylglycinyl)phosphorochloridate (0.68 g, 1.64mmol) and TEA (0.66 mL, 4.75 mmol) as a white solid, which was furtherrepurifed by preparative HPLC (0.14 g, 10%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-9.79 (d, J=22.6 Hz),−9.82 (d, J=22.6Hz), −12.17 (d, J=21.20 Hz), −12.20 (d, J=21.0 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.38-8.34 (2H, m, H-Ar), 8.24 (0.5H, d,⁵J_(H-F)=2.0 Hz, H-8), 8.22 (0.5H, d, ⁵J_(H-F)=2.0 Hz, H-8), 7.85-7.83(2H, m, H-Ar), 7.65-7.60 (3H, m, H-Ar), 7.48-7.44 (3H, m, H-Ar),7.38-7.29 (2H, m, H-Ar), 6.39 (0.5H, t, J=1.5 Hz, H-1′), 6.36 (0.5H, t,J=1.5 Hz, H-1′), 5.17-5.15 (0.5H, m, H-2′), 5.14-5.07 (2H, m, OCH₂Ph),5.05-5.02 (0.5H, m, H-2′), 4.57-4.50 (1H, m, H-3′), 4.27-4.24 (2H, m,H-5′), 4.15-4.10 (1H, m, H-4′), 3.05 (6H, q, J=7.0 Hz, N(CH₂CH₃)₃), 1.90(6H, s, NHC(CH₃)₂), 1.31 (9H, t, J=7.5 Hz, N(CH₂CH₃)₃).

¹³C NMR (125 MHz, CD₃OD): δ_(C) 176.53, 174.95 (2×d, ³J_(C-P)=6.7 Hz,C=0, ester), 156.64 (C-6), 154.08 (C-2), 150.16, 149.36 (2×d,³J_(C-P)=7.0 Hz, C-Ar), 146.91, 146.85 (C-4), 142.89 (C-8), 136.23,135.99, 134.79 (C-Ar), 128.07, 127.70, 127.65, 127.61, 127.16, 126.99,126.18, 125.83, 125.59, 125.43, 125.06, 124.76, 124.20, 122.57, 122.25,122.20, 121.44 (CH—Ar), 117.07 (C-5), 115.9 (d, ⁴J_(C-P)=3.2 Hz, CH—Ar),113.15 (d, ⁴J_(C-P)=3.2 Hz, CH—Ar), 94.98, 94.95 (2×d, ¹ J_(C-F)=191.5Hz, C-2′), 82.80 (d, ³J_(C-F)=3.8 Hz C-4′), 82.68 (d, ²J_(C-F)=24.5 HzC-1′), 73.57, 73.45 (2 × d, ²J_(C-F)=24.5 Hz, C-3′), 66.69, 66.37(OCH₂Ph), 64.94 (C-5′), 56.95, 55.87 (NHC(CH₃)₂), 46.28 (N(CH₂CH₃)₃),26.10 (d, ³J_(C-P)=4.1 Hz, NHC(CH₃)₂), 7.86 (N(CH₂CH₃)₃).

C₃₁₁₁₃₂ClFN₆O₁₀P₂mass required m/z 764.13; found MS (ES⁻) m/z: 763.15([M-H]⁻)

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=14.85 min.

Example 12:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[phenyl(cyclohexoxy-L-valinyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.96 g, 1.64 mmol),phenyl(cyclohexoxy-L-valinyl)phosphorochloridate (0.61 g, 1.64 mmol) andTEA (0.66 mL, 4.75 mmol) as a white solid (0.19 g, 14%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-6.74 (d, J=21.2 Hz), −6.78 (d, J=21.0Hz), −12.0 (d, J=21.4 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.18 (0.5H, d, ⁵J_(H-F)=1.0 Hz, H-8),8.17 (0.5H, d, ⁵J_(H-F)=1.0 Hz, H-8), 7.22-7.15 (4H, m, H-Ar), 7.01-6.98(1H, m, H-Ar), 6.34 (0.5H, t, J=1.5 Hz, H-1′), 6.30 (0.5H, t, J=1.5 Hz,H-1′), 5.08-5.06 (0.5H, m, H-2′), 4.97-4.96 (0.5H, m, H-2′), 4.62-4.54(1H, m, OCH), 4.46 (0.5H, apparent t, J=2.0 Hz, H-3′), 4.43 (0.5H,apparent t, J=2.0 Hz, H-3′), 4.15-4.12 (2H, m, H-5′), 4.06-4.02 (1H, m,H-4′), 3.68 (0.5H, apparent dd, J=10.0 Hz, J=5.5 Hz, NHCHCH(CH₃)₂), 3.64(0.5H, apparent dd, J=10.0 Hz, J=5.5 Hz, NHCHCH(CH₃)₂), 3.05 (6H, q,J=7.0 Hz, N(CH₂CH₃)₃), 1.91-1.80 (1H, m, NHCHCH(CH₃)₂), 1.67-1.58 (5H,m, CH₂), 1.39-1.37 (1H, m, CH₂), 1.36-1.31 (3H, m, CH₂), 1.31 (9H, t,J=7.5 Hz, N(CH₂CH₃)₃), 0.86-0.78 (6H, m, NHCHCH(CH₃)₂).

¹³C NIVIR (125 MHz, CD₃OD): δ_(C) 171.98, 171.95 (2×d, ³J_(C-P)=4.5 Hz,C═O, ester), 156.69 (C-6), 154.14 (C-2), 151.02 (d, ²J_(C-P)=3.8 Hz,C-Ar), 150.23 (C-4), 142.25 (C-8), 129.16, 128.60, 124.56, 124.47,120.44 (CH—Ar), 117.13 (C-5), 94.96 (d, =191.4Hz, C-2′), 82.90 (d,³J_(C-F)=3.8 Hz C-4′), 82.81 (d, ²J_(C-F)=32.5 Hz C-1′), 73.59, (d,²J_(C-F)=24.5 Hz, C-3′), 73.42 (d, ²J_(C-F)=16.8 Hz, C-3′), 62.88 (d,²J_(C-P)=7.5 Hz, C—C-5′), 60.45, 60.20 (NHCHCH(CH₃)₂), 46.42(N(CH₂CH₃)₃), 32.56, 32.09 (NHCHCH(CH₃)₂), 31.14 (CH₂), 25.00 (CH₂),23.25 (CH₂), 17.79, 16.76 (NHCHCHCH3), 7.82 (N(CH₂CH₃)₃).

C₂₇H₃₆ClFN₆O₁₀P₂ mass required m/z 720.16; found MS (ES⁻) m/z: 719.17([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=15.73 min.

Example 13:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-naphthyl(neopentoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.57 g, 0.97 mmol),1-naphthyl(neopentoxy-L-alaninyl)phosphorochloridate (0.37 g, 0.97 mmol)and TEA (0.39 mL, 2.86 mmol) as a white solid (0.13 g, 16%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-7.41 (d, J=23.5 Hz), −7.82 (d, J=22.5Hz), −11.82 (d, J=22.69 Hz), −11.95 (d, J=21.80 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.36-8.25 (2H, m, H-Ar), 7.85-7.79 (2H,m, H-Ar), 7.68-7.65 (2H, m, H-8), 7.58-7.36 (2H, m, H-Ar), (6H, m,H-Ar), 6.4 (1H, dd J=16.68 Hz, 4.1 Hz, H-1′), 5.19-5.05 (1H, m, H-2′),4.58-4.53 (1H m, H-3′), 4.29-4.25 (2H, m, H-5′), 4.17-4.14 (1.5H, m,H-4′ and NHCHCH₃), 4.06-4.01 (0.5H, m, NHCHCH₃), 3.75-3.66 (2H, m,OCH₂C(CH₃)₃), 3.14 (6H, q, J=7.0 Hz, N(CH₂CH₃)₃), 1.31-1.26 (12H, m,NHCHCH₃, N(CH₂CH₃)₃).

C₂₈H₃₄ ClFN₆O₁₀P₂ mass required m/z 730.15; found MS (ES⁻) m/z: 729.09([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB (pH 7.2)/AcCN from 90/10 to0/100 in 30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomerswith t_(R)=14.63 min.

Example 14:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[phenyl(neopentoxy-D-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),phenyl(neopentoxy-D-alaninyl)phosphorochloridate (0.49 g, 1.48 mmol) andTEA (0.59 mL, 4.25 mmol) as a white solid (0.16 g, 14%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-7.68 (d, J=21.0 Hz), −7.91 (d, J=21.0Hz), −12.04 (d, J=20.08 Hz), −12.14 (d, J=20.08 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.30 (0.5H, d, J_(H-F)=2.0 Hz, H-8), 8.29(0.5H, d, J_(H-F)=2.0 Hz, H-8), 7.35-7.27 (4H, m, H-Ar), 7.18-7.14 (1H,m, H-Ar), 6.45 (0.5H, t, J=3.5 Hz, H-1′), 6.42 (0.5H, t, J=3.5 Hz,H-1′), 5.20 (0.5H, apparent t, J=3.5 Hz, H-2′), 5.09 (0.5H, apparent t,J=3.5 Hz, H-2′), 4.61-4.54 (1H, m, H-3′), 4.27-4.23 (2H, m, H-5′),4.17-4.10 (2H, m, NHCHCH₃, H-4′), 3.80-3.72 (2H, m, OCH₂C(CH₃)₃), 3.33(6H, q, J=7.0 Hz, N(CH₂CH₃)₃), 1.42 (1.5H, dd, J=7.0 Hz, J=0.5 Hz,NHCHCH₃), 1.36 (1.5H, dd, J=7.0 Hz, J=0.5 Hz, NHCHCH₃), 1.31 (9H, t,J=7.5 Hz, N(CH₂CH₃)₃), 0.80 (9H, s, OCH₂C(CH₃)₃).

¹³C NMR (125 MHz, CD₃OD): δ_(C) 173.56, 173.48 (apparent t, ³J_(C-P)=7.6Hz, C═O, ester), 156.70 (C-6), 154.13 (C-2), 150.89 (apparentt,²J_(C-P)=6.6 Hz, C-Ar), 150.24 (C-4), 142.68 (C-8), 129.22, 128.67,124.65, 124.60, 122.34 (CH—Ar), 120.38, 120.36 (2×d, ³J_(C-P)=4.6 Hz,CH—Ar), 117.14

(C-5), 95.0, 94.99 (2×d, ¹1 C—F=191.6 Hz, C-2′), 82.76 (d, ³J_(C-F)=8.25Hz, C-4′), 82.68 (d, ²J_(C-F) =8.5 Hz, C-1′), 73.95, 73.92(OCH₂C(CH₃)₃), 73.58, 73.50 (2×d, ²J_(C-F)=24.5 Hz, C-3′), 64.98, 64.86(2×d, ²J_(C-P)=4.6 Hz, C-5′), 50.36, 50.27 (2×d, ³J_(C-P)=2.4 Hz,NHCHCH₃), 46.36 (N(CH₂CH₃)₃), 30.92, 30.89 (OCH₂C(CH₃)₃), 25.35, 25.33(OCH₂C(CH₃)₃), 19.75, 19.61 (2×d, ³J_(C-P)=5.6 Hz, NHCHCH₃), 7.82(N(CH₂CH₃)₃).

C₂₄H₃₂ClFN₆O₁₀P₂mass required m/z 680.94; found MS (ES) m/z: 679.19([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=14.23 min.

Example 15:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5phenyl(isopropoxy-L-alaninyl)J diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),phenyl(isopropoxy-L-alaninyl)phosphorochloridate (0.45 g, 1.48 mmol) andTEA (0.59 mL, 4.25 mmol) as a white solid (0.126 g, 11%).

³¹P NMR (202 MHz, CD₃OD): δ_(P) −7.62 (d, J=21.0 Hz), −7.85 (d, J=21.0Hz), −12.14 (apparent t, J=20.2 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.30 (1H, d, ⁵J_(H-F)=2.0 Hz, H-8),7.35-7.27 (4H, m, H-Ar), 7.18-7.14 (1H, m, H-Ar), 6.45 (0.5H, t, J=3.5Hz, H-1′), 6.42 (0.5H, t, J=3.5 Hz, H-1′), 5.22 (0.5H, q, J=3.5 Hz,H-2′), 5.13 (0.5H, q, J=3.5 Hz, H-2′), 4.96-4.94 (1H, m, OCH(CH₃)₂),4.61 — 4.58 (0.5H, m, H-3′), 4.57 — 4.55 (0.5H, m, H-3′), 4.29 — 4.25(2H, m, H-5′), 4.19 — 4.15 (1H, m, NHCHCH₃), 4.07-4.00 (1H, m, H-4′),3.33 (6H, q, J=7.0 Hz, N(CH₂CH₃)₃), 1.38 (1H, d, J=7.5 Hz, NHCHCH₃),1.33 (2H, dd, J=7.5 Hz, NHCHCH₃), 1.31 (9H, t, J=7.5 Hz, N(CH₂CH₃)₃),1.20 (6H, apparent t, J=6.0 Hz, OCH(CH₃)₂).

¹³C NMR (125 MHz, CD₃OD): & 173.10 (d, ³J_(C-P)=7.1 Hz, C═O, ester),173.02 (C═O, ester), 156.73 (C-6), 154.16 (C-2), 150.92, 150.86 (2×d,²J_(C-P)=5.2 Hz, C-Ar), 150.24 (C-4), 142.55 (C-8), 129.26, 124.64,124.62 (CH—Ar), 120.34 (d, ³J_(C-P)=4.5 Hz, CH—Ar), 117.16 (C-5), 95.00(d, ¹J_(C-F)=191.5 Hz, C-2′), 82.90 (d, ³J_(C-F)=6.5 Hz C-4′), 82.80,82.76 (2×d, ²J_(C-F)=10.5 Hz C-1′), 73.60, 73.59 (2×d, ²J_(C-F)=24.5 HzC-3′), 68.71 (OCH(CH₃)₂), 65.08 (d,²J_(C-P)=4.2 Hz, C-5′), 50.39, 50.34(2×d, ²J_(C-P)=1.8 Hz, NHCHCH₃), 46.23 (N(CH₂CH₃)₃), 20.61, 20.54(OCH(CH₃)₂), 19.60, 19.47 (2×d, ³J_(C-P)=6.0 Hz, NHCHCH₃), 7.86(N(CH₂CH₃)₃).

C₂₂H₂₈ClFN₆O₁₀P₂ mass required m/z 652.10; found MS (ES⁻) m/z: 651.13([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=12.32 min.

Example 16:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-phenyl(methoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),phenyl(methoxy-L-alaninyl)phosphorochloridate (0.41 g, 1.48 mmol) andTEA (0.59 mL, 4.25 mmol) as a white solid (0.182 g, 17%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-7.73 (d, J=20.7 Hz), −7.84 (d, J=20.8Hz), −12.04 (d, J=21.0 Hz), −12.11 (d, J=20.8 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.31 (0.5H, d, J_(H-F)=2.5 Hz, H-8), 8.30(0.5H, d, J_(H-F)=2.0 Hz, H-8), 7.35-7.27 (4H, m, H-Ar), 7.18-7.14 (1H,m, H-Ar), 6.44 (0.5H, dd, J_(H-F)=16.4 Hz, J=3.9 Hz, H-1′), 6.42 (0.5H,dd, J_(H-F)=16.4 Hz, J=3.7 Hz, H-1′), 5.20-5.09 (1H, m, H-2′), 4.68-4.52(1H, m, H-3′), 4.28-4.24 (2H, m, H-5′), 4.18-4.15 (1H, m, H-4′),4.12-4.07 (1H, m, NHCHCH₃), 3.66 (1.5H, s, OCH₃), 3.65 (1.5H, s, OCH₃),3.78 (6H, q, J=7.0 Hz, N(CH₂CH₃)₃), 1.37 (1.5H, dd, J=7.0 Hz, J=0.5 Hz,NHCHCH₃), 1.32 (1.5H, dd, J=7.0 Hz, J=0.5 Hz, NHCHCH₃), 1.14 (9H, t,J=7.5 Hz, N(CH₂CH₃)₃).

C₂₀H₂₄ClFN₆O₁₀P₂ mass required m/z 724.07; found MS (ES⁻) m/z: 723.01([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=12.24 min.

Example 17:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-1-naphthyl(benzoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),1-naphthyl(benzoxy-L-alaninyl)phosphorochloridate (0.59 g, 1.48 mmol)and TEA (0.59 mL, 4.25 mmol) as a white solid (0.226 g, 18%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-7.26 (d, J=20.2 Hz), −7.61 (d, J=20.2Hz), −12.07 (d, J=20.40 Hz), −12.27 (d, J=20.40 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.19-8.17 (2H, m, H-Ar), 8.13 (0.5H, d,J_(H-F)=2.0 Hz, H-8), 8.12 (0.5H, d, J_(H-F)=2.0 Hz, H-8), 7.72-7.68(2H, m, H-Ar), 7.54-7.52 (1H, apparent d, J=9.5 Hz, H-Ar), 7.44-7.40(4H, m, H-Ar), 7.37-7.35 (1H, m, H-Ar), 6.28 (0.5H, t, J=4.0 Hz, H-1′),6.25 (0.5H, t, J=4.0 Hz, H-1′), 5.06-5.02 (0.5H, m, H-2′), 4.95-4.93(0.5H, m, H-2′), 4.89-4. 80 (2H, OCH₂Ph), 4.43-4.43 (0.5H, m, H-3′),4.41-4.40 (0.5H, m, H-3′), 4.15-4.12 (2H, m, H-5′), 4.09-4.06 (1H, m,NHCHCH₃), 4.03-4.01 (1H, m, H-4′), 3.33 (6H, q, J=7.0 Hz, N(CH₂CH₃)₃),1.23 (1.5H, dd, J=7.0 Hz, J=0.5 Hz, NHCHCH₃), 1.20 (1.5H, dd, J=7.0 Hz,J=0.5 Hz, NHCHCH₃), 1.31 (9H, t, J=7.5 Hz, N(CH₂CH₃)₃).

C₃₀H₃₀C₁FN₆O₁₀P₂ mass required m/z 750.99; found MS (ES⁻) m/z: 749.19([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, t_(R)=14.69, 14.81 min.

Example 18:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-phenyl(hexoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),1-phenyl(hexoxy-L-alaninyl)phosphorochloridate (0.51 g, 1.48 mmol) andTEA (0.59 mL, 4.25 mmol) as a white solid (0.235 g, 20%).

³¹P NMR (202 MHz, CD₃OD): Sr −7.55 (d, J=23.00 Hz), −7.77 (d, J=19.40Hz), −12.09 (d, J=19.40 Hz), −12.19 (d, J=20.10 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) H 8.18 (0.5H, d, J_(H-F)=2.0 Hz, H-8),8.17 (0.5H, d, J_(H-F)=2.0 Hz, H-8), 7.23-7.11 (4H, m, H-Ar), 7.08-7.03(1H, m, H-Ar), 6.33 (0.5H, dd, J=4.0, 1.5 Hz, H-1′), 6.29 (0.5H, dd,J=4.0 Hz, J=1.5 Hz, H-1′), 5.08-5.06 (0.5H, m, H-2′), 4.98-4.96 (0.5H,m, H-2′), 4.47-4.45 (0.5H, m, H-3′), 4.44-4.42 (0.5H, m, H-3′),4.15-4.11 (2H, m, H-5′), 4.06-4.03 (1H, m, NHCHCH₃), 3.97-3.90 (5H, m,H-4′, 2 ×CH₂, OCH₂(CH₂)₄CH₃), 3.84-3.78 (1H, m, OCH₂(CH₂)₄CH₃), 3.33(6H, q, J=7.0 Hz, N(CH₂CH₃)3, 1.50-1.43 (4H, m, 2 ×CH₂, OCH₂(CH₂)₄CH₃),1.31-1.22 (6H, m, CH₂, OCH₂(CH₂)₄CH₃, NHCHCH₃), 1.31 (9H, t, J=7.5 Hz,N(CH₂CH₃)₃), 0.81-0.76 (3H, m, OCH₂(CH₂)₄CH₃).

C₂₅H₃₄ClFN₆O₁₀P₂ mass required m/z 694.97; found MS (ES⁻) m/z: 693.14([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, t_(R)=15.13 and 15.21 min.

Example 19:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-5,6,7,8-tetrahydro-1-naphthyl(benzoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),5,6,7,8-tetrahydro-1-naphthyl(benzoxy-L-alaninyl)phosphorochloridate(0.603 g, 1.48 mmol) and TEA (0.59 mL, 4.25 mmol) as a white solid (0.19g, 15%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-7.06 (d, J=24.0 Hz), −7.55 (d, J=21.2Hz), −11.24 (d, J=21.2 Hz), −11.38 (d, J=24.0 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.27 (0.5H, d, J_(H-F)=2.0 Hz, H-8), 8.25(0.5H, d, J_(H-F)=2.0 Hz, H-8), 7.33-7.30 (3H, m, H-Ar), 7.22-7.18 (2H,m, H-Ar), 7.00-6.93 (2H, m, H-Ar), 6.84 (1H, apparent t, J=6.5 Hz,H-Ar), 6.72 (1H, apparent d, J=7.5 Hz, H-Ar), 6.43 (0.5H, dd, J=4.0 Hz,J =2.0 Hz, H-1′), 6.39 (0.5H, dd, J =4.0 Hz, J=2.0 Hz, H-1′), 5.19-5.16(0.5H, m, H-2′), 5.09-5.06 (0.5H, m, H-2′), 5.06-5.03 (2H, m, OCH₂Ph),4.56-4.49 (1H, m, H-3′), 4.27-4.10 (3H, m, H-5′, H-4′), 4.02-3.97 (1H,m, NHCHCH₃), 3.33 (6H, q, J=7.0 Hz, N(CH₂CH₃)₃), 2.73 (4H, bt, 2 ×CH₂),1.75-1.72 (4, m, 2 ×CH₂), 1.42 (1.5H, d, J=7.5 Hz, NHCHCH₃), 1.38 (1.5H,dd, J=7.0 Hz, NHCHCH₃), 1.31 (9H, t, J=7.5 Hz, N(CH₂CH₃)₃).

C₃₀H₃₄ClFN₆O₁₀P₂ mass required m/z 754.15; found MS (ES⁻) m/z: 753.23([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, t_(R)=15.32 and 15.44 min.

Example 20:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-ethyl(benzoxy-L-alaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),ethyl(benzoxy-L-alaninyl)phosphorochloridate (0.45 g, 1.48 mmol) and TEA(0.59 mL, 4.25 mmol) as a white solid (0.218 g, 20%).

³¹P NMR (202 MHz, CD₃OD): δ_(P)-2.73 (d, J=20.2 Hz), −3.04 (d, J=20.4Hz), −11.96 (d, J=20.2 Hz), −12.07 (d, J=20.04 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.32 (0.5H, d, J_(H-F)=2.0 Hz, H-8), 8.31(0.5H, d, J_(H-F)=2.0 Hz, H-8), 7.37-7.27 (5H, m, H-Ar), 6.46 (0.5H, dd,J=4.0 Hz, J=2.0 Hz, H-1′), 6.43 (0.5H, dd, J=4.0 Hz, J=2.0 Hz, H-1′),5.24-5.22 (0.5H, m, H-2′), 5.20-5.14 (2H, m, OCH₂Ph), 5.13-5. 11 (0.5H,m, H-2′), 4.62-4.60 (0.5H, m, H-3′), 4.59-4.57 (0.5H, m, H-3′),4.29-4.26 (2H, m, H-5′), 4.19-4.17 (2H, m, NHCHCH₃), 4.12-4.04 (3H, m,H-4′, OCH₂CH₃), 2.86 (6H, q, J=7.0 Hz, N(CH₂CH₃)₃), 1.43 (1.5H, dd,J=7.0 Hz, J=0.5 Hz, NHCHCH₃), 1.42 (1.5H, dd, J=7.0 Hz, J=0.5 Hz,NHCHCH₃), 1.27-1.23 (3H, m, OCH₂CH₃), 1.19 (9H, t, J=7.5 Hz,N(CH₂CH₃)₃).

C₂₂H₂₈ClFN₆O₁₀P₂mass required m/z 652.89; found MS (ES⁻) m/z: 651.15([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=14.12 min.

Example 21:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′phenyl(benzoxy-L-leucinylil diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-(3-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),phenyl(benzoxy-L-leucinyl)phosphorochloridate (0.58 g, 1.48 mmol) andTEA (0.59 mL, 4.25 mmol) as a white solid (0.30 g, 24%).

³¹P NMR (202 MHz, CD₃OD): δ_(P) −7.28 (d, J=21.4 Hz), −7.57 (d, J=21.6Hz), −12.18 (d, J=21.6 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.18 (1H, s, H-8), 7.19-7.10 (8H, m,H-Ar), 7.06-6.99 (2H, m, H-Ar), 6.32 (0.5H, t, J=3.5 Hz, H-1′), 6.29(0.5H, t, J=3.5 Hz, H-1′), 5.10-5.08 (0.5H, m, H-2′), 4.97-4.88 (2.5H,m, H-2′, OCH₂Ph), 4.46 (0.5H, apparent t, J=3.5 Hz, H-3′), 4.43 (0.5H,apparent t, J=3.5 Hz, H-3′), 4.17-4.13 (2H, m, H-5′), 4.06-4.04 (1H, m,H-4′), 3.94-3.88 (1H, m, NHCHCH₂CH(CH₃)₂), 3.33 (6H, q, J=7.0 Hz,N(CH₂CH₃)₃), 1.13 (9H, t, J=7.5 Hz, N(CH₂CH₃)₃), 1.43-1.37 (3H, m,NHCHCH₂CH(CH₃)₂), 0.75-0.72 (3H, m, NHCHCH₂CH(CH₃)₂), 0.65-0.64 (3H,apparent dd, J=14.0 Hz, J=6.0 Hz, NHCHCH₂CH(CH₃)₂).

¹³C NMR (125 MHz, CD₃OD): δ_(C) 173.43, 173.22 (2×d, ³J_(C-P)=3.5 Hz,C═O, ester), 156.78, 156.72 (C-6), 154.16 (C-2), 150.92 (d, ²J_(C-P)=7.0Hz, C-Ar), 150.24 (C-4), 135.84, 84, 135.81 (C-8), 129.26, 128.74,128.26, 128.13, 127.95, 127.90, 127.88, 127.83, 124.68, 124.57, 122.30(CH—Ar), 120.44 (d, ³J_(C-P)=4.6 Hz, CH—Ar), 120.27 (d, ³J_(C-P)=5.2 Hz,CH—Ar), 119.98, 119.94 (CH—Ar), 117.17 (C-5), 95.00 (d, ¹ J_(C-F)=191.6Hz, C-2′), 82.90 (d, ³J_(C-F)=4.1 Hz C-4′), 82.74 (d, ²J_(C-F) =11.0 HzC-1′), 73.63, 73.58 (2×d, ²J_(C-F)=24.5 Hz, C-3′), 66.43, 66.05(OCH₂Ph), 65.10 (d, ²J_(C-P)=5.5 Hz, C-5′), 53.70 (NHCH), 53.31 (d,²J_(C-P)=5.5 Hz, NHCH), 46.21 (N(CH₂CH₃)₃), 43.20, 43.00 (2 x=7.0 Hz,NHCHCH₂), 24.13, 24.08 (NHCHCH₂CH(CH₃)₂), 21.66, 21.45, 21.07, 20.99(NHCHCH₂CH(CH₃)₂), 7.83 (N(CH₂CH₃)₃).

C₂₉H₃₄ClFN₆O₁₀P₂ mass required m/z 742.15; found MS (ES) m/z: 741.18([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=14.91 min.

Example 22:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-phenyl(benzoxy-L-phenylalaninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),phenyl(benzoxy-L-phenylalaninyl)phosphorochloridate (0.63 g, 1.48 mmol)and TEA (0.59 mL, 4.25 mmol) as a white solid (0.185 g, 14%).

³¹P NMR (202 MHz, CD₃OD): δ_(P) −7.88 (d, J=20.6 Hz), −7.98 (d, J=20.0Hz), −12.00 (d, J=21.21 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.17 (0.5H, d, ⁵J_(H-F)=2.5 Hz, H-8),8.14 (0.5H, d, ⁵J_(H-F)=2.5 Hz, H-8), 7.18-7.08 (10, m, H-Ar), 6.97-6.87(5H, m, H-Ar), 6.30 (0.5H, dd, J=4.0 Hz, J=1.5 Hz, H-1′), 6.27 (0.5H,dd, J=4.0 Hz, J=1.5 Hz, H-1′), 5.08-5.05 (0.5H, m, H-2′), 4.97-4.95(0.5H, m, H-2′), 4.86-4.77 (4H, m OCH₂Ph, NHCHCH₂Ph), 4.46-4.44 (0.5H,m, H-3′), 4.43-4.41 (0.5H, m, H-3′), 4.25-4.17 (1H, m, NHCHCH₂Ph),4.16-4.12 (2H, m, H-5′), 4.10-4.05 (1H, m, H-4′), 3.33 (6H, q, J=7.0 Hz,N(CH₂CH₃)₃), 1.31 (9H, t, J=7.5 Hz, N(CH₂CH₃)₃).

¹³C NMR (125 MHz, CD₃OD): δ_(C) 173.46 (d, ³J_(C-P)=4.6 Hz, C=0, ester),172.05 (d, ³J_(C-P)=4.5 Hz, C═O, ester), 156.68 (C-6), 154.13 (C-2),153.36 (d, ²J_(C-P)=7.1 Hz, C-Ar), 150.85, 150.83 (C-4), 150.23 (C-Ar),136.73, 136.11 (d, ²J_(C-P)=4.7 Hz, C-Ar), 135.69 (C-8), 135.52, 135.50(C-Ar), 129.20 (d, ³J_(C-P)=3.1 Hz, CH—Ar), 128.74, 128.09, 128.05 (d,³J_(C-P)=2.8 Hz, CH—Ar), 127.90, 127.84, 127.78, 126.44, 126.25, 124.59,122.40, 120.38, 120.34, 120.30, 120.26, 120.15, 120.11 (CH—Ar), 117.15(C-5), 95.01 (d, ¹ J_(C-F)=191.0 Hz, C-2′), 95.00 (d, ¹ J_(C-F)=191.0Hz, C-2′), 82.84 (d, ³J_(C-F)=3.8 Hz C-4′), 82.68 (d, ²J_(C-F)=32.5 HzC-1′), 73.58 (d, ²J_(C-F)=24.5 Hz C-3′), 66.45, 66.44 (OCH₂Ph), 66.11(OCH₂Ph), 65.04 (d, ²J_(C-P)=4.8 Hz, C-5′), 56.44 (NHCHCH₂Ph), 56.19(NHCHCH₂Ph), 46.21 (N(CH₂CH₃)₃), 40.79, 40.74 (CH₂Ph), 9.85(N(CH₂CH₃)₃).

C₃₂H₃₂ClFN₆O₁₀P₂mass required m/z 776.13; found MS (ES) m/z: 775.17([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=24.28 min.

Example 23:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-phenyl(benzoxy-L-prolinyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),phenyl(benzoxy-L-prolinyl)phosphorochloridate (0.55 g, 1.48 mmol) andTEA (0.59 mL, 4.25 mmol) as a white solid (0.17 g, 14%).

³¹P NMR (202 MHz, CD₃OD): δ_(P) −9.05 (d, J=17.6 Hz), −9.72 (d, J=21.6Hz), −11.75 (d, J=17.6 Hz), −12.12 (d, J=21.6 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) H 8.17 (0.5H, d, J_(H-F)=2.5 Hz, H-8),8.16 (0.5H, d, J_(H-F)=2.5 Hz, H-8), 7.21-7.15 (8H, m, H-Ar), 7.11-7.09(1H, m, H-Ar), 7.04-7.00 (1H, m, H-Ar), 6.32 (0.5H, d, J=4.0 Hz, H-1′),6.31 (0.5H, d, J=4.0 Hz, H-1′), 5.08-5.06 (0.5H, m, H-2′), 5.01-4.99(0.5H, m, H-2′), 4.98-4.91 (2H, m, OCH₂Ph), 4.48-4.45 (1H, m, NCH),4.43-4.41 (0.5H, m, H-3′), 4.34-4.30 (0.5H, m, H-3′), 4.16-4.09 (2H, m,H-5′), 4.05-4.00 (1H, m, H-4′), 3.46-3.38 (2H, m, NCH2), 2.21-2.05 (1H,m, one H of NCH2CH₂), 1.85-1.76 (3H, m, 1×H, NCH2CH_(2, 2)×H,NCH2CH₂CH₂), 3.33 (6H, q, J=7.0 Hz, N(CH₂CH₃)3, 1.31 (9H, t, J=7.5 Hz,N(CH₂CH₃)₃).

¹³C NMR (125 MHz, CD₃OD): δ_(C) 173.60, 173.31 (C═O, ester), 156.69(C-6), 154.14 (C-2), 150.78 (d, ²J_(C-P)=3.8 Hz, C-Ar), 150.22 (C-4),136.37 (C-8), 136.03, 135.96 (C-Ar), 129.29, 129.23, 128.13, 127.80,127.76, 127.67, 124.57 (CH—Ar), 120.15 (d, ³J_(C-P)=5.2 Hz, CH—Ar),119.96 (d, ³J_(C-P)=5.2 Hz, CH—Ar), 117.14 (C-5), 95.02 (d, ¹J_(C-F)=191.7 Hz, C-2′), 94.99 (d, ¹ J_(C-F)=191.7 Hz, C-2′), 82.85 (d,³J_(C-F)=5.6 Hz C-4′), 82.71 (d, ²J_(C-F)=32.5 Hz, C-1′), 73.59 (d,²J_(C-F)=24.5 Hz, C-3′), 73.55 (d, ²J_(C-F)=24.5 Hz, C-3′), 66.32, 66.25(OCH₂Ph), 65.02 (d, ²J_(C-P)=4.0 Hz, C-5′), 64.90 (d, ²J_(C-P)=4.0 Hz,C-5′), 60.72 (²J_(C-P)=7.75 Hz, NCH), 60.35 (²J_(C-P)=7.75 Hz, NCH),46.31 (N(CH₂CH₃)₃), 30.99 (²J_(C-P)=11.0 Hz, NCH2), 30.89 (²J_(C-P)=11.0Hz, NCH2), 24.84 (³J_(C-P)=9.0 Hz, NCH2CH₂), 24.40 (³J_(C-P)=9.0 Hz,NCHCH2), 7.85 (N(CH₂CH₃)₃).

C₂₈H₃₀C₁FN₆O₁₀P₂ mass required m/z 726.97; found MS (ES⁻) m/z: 725.67([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=15.39 min.

Example 24:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-phenyl(methoxy-L-metioninyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),phenyl(methoxy-L-methioninyl)phosphorochloridate (0.49 g, 1.48 mmol) andTEA (0.59 mL, 4.25 mmol) as a white solid (0.25 g, 22%).

³¹P NMR (202 MHz, CD₃OD): δ_(P) −7.41 (d, J=21.33 Hz), −7.69 (d, J=21.6Hz), −12.29 (d, J=20.6 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) H 8.19 (0.5H, d, J_(H-F)=2.1 Hz, H-8),8.18 (0.5H, d, J_(H-F)=2.0 Hz, H-8), 7.21-7.16 (4H, m, H-Ar), 7.09-7.02(1H, m, H-Ar), 6.32 (0.5H, dd, J_(H-F)=16.6 Hz, J=4.3 Hz, H-1′), 6.31(0.5H, dd, J_(H-F)=16.6 Hz, J=4.2 Hz, H-1′), 5.02 (0.5H, dd J_(H-F)=51.9Hz, A-F=2.2 Hz, H-2′) 5.00 (0.5H, dd, J_(H-F)=51.9 Hz, JH-H=2.2 Hz,H-2′), 4.48-4.43 (1H, m, H-3′), 4.17-4.13 (2H, m, H-5′), 4.08-4.00 (m,2H, NHCHCH₃, H-4′), 3.56, 3.43 (3H, 2s, OCH₃), 2.80 (6H, q, J=7.0 Hz,N(CH₂CH₃)3, 2.42-2.39 (1H, m, CH₃SCH2CH₂a), 2.28-2.24 (1H, m,CH₃SCH2CH₂b), 1.90, 1.86 (3H, 2 ×s, SCH3), 1.90-1.71 (2H, m,CH₃SCH2CH₂a).

C₂₂H₂₈ClFN₆O₁₀P₂S mass required 684.96; found MS (ES-) m/z: 683 ([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=12.28 min.

Example 25:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-phenyl(ethoxy-L-isoleucinyl)]diphosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),phenyl(ethoxy-L-isoleucinyl)phosphorochloridate (0.49 g, 1.48 mmol) andTEA (0.59 mL, 4.25 mmol) as a white solid (0.26 g, 23%).

³¹P NMR (202 MHz, CD₃OD): δ_(P) −6.84 (d, J=21.5 Hz), −6.99 (d, J=22.3Hz), −12.00 (d, J=21.6 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.30, (0.5H, d, J_(H-F)=2.4 Hz, H-8),8.29 (0.5H, d, J_(H-F)=2.3 Hz, H-8), 7.35-7.23 (4H, m, H-Ar), 7.19-7.13(1H, m, H-Ar), 6.44 (0.5H, dd, J_(H-F)=16.7 Hz, J=3.9 Hz, H-1′), 6.43(0.5H, dd, J_(H-F)=16.7 Hz, J_(H-H)=4.2 Hz, H-1′), 5.21-5.08 (1H, m,H-2′), 4.59-4.54 (1H, m, H-3′), 4.28-4.25 (2H, m, OCH₂CH₃), 4.18-4.07(3H, m, H-5′a, H-4′, NHCHCH₃), 3.83 (0.5H, dd, J=9.8 Hz, J=5.7 Hz, H-5′b,), 3.82 (0.5H, dd, J=9.8 Hz, J=5.7 Hz, Hb-5′), 3.33 (6H, q, J=7.0 Hz,N(CH₂CH₃)3, 1.77-1.68 (1H, m, CHCH3), 1.55-1.42 (1H, m, CHCH2a),1.15-1.09 (1H, m, CHCH2b), 1.27-1.20 (12H, m, N(CH₂CH₃)₃, OCH₂CH₃),0.91-0.82 (6H, CHCH3, CH₂CH₃).

C₂₄H₃₂ClFN₆O₁₀P₂ mass required 680.14; found MS (ES-) m/z: 680.18([M—H]⁻)

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=13.57 min.

Example 26:2-Chloro-9-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine-5′-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphorothiol-phosphate triethylammonium salt

Prepared according to the general procedure from2-chloro-9-(2′-deoxy-2′-fluoro-(3-D-arabinofuranosyl)adenine-5′-monophosphateTEA salt (0.86 g, 1.48 mmol),1-naphthyl(benzoxy-L-alaninyl)phosphorothiolchloridate (0.62 g, 1.48mmol) and TEA (0.59 mL, 4.25 mmol) as a white solid (0.054 g, 4%).

³¹P NMR (202 MHz, CD₃OD): Sr 55.05 (d, J=28.5 Hz), 53.71 (d, J=28.5 Hz),−12.24 (broad d, J=28.7 Hz).

¹H NMR (500 MHz, CD₃OD): δ_(H) 8.37-8. 34 (1H, m, H-Ar), 8.28 (0.5H, d,J_(H-F)=2.0 Hz, H-8), 8.26 (0.5H, d, J_(H-F)=2.0 Hz, H-8), 7.85-7.82(1H, m, H-Ar), 7.66-7.64 (2H, m, H-Ar), 7.50-7.43 (2H, m, H-Ar),7.38-7.19 (5H, m, H-Ar), 6.42 (0.5H, t, J=4.0 Hz, H-1′), 6.39 (0.5H, t,J=4.0 Hz, H-1′), 5.19-5.16 (1H, m, H-2′), 5.11-5.02 (2H, m, OCH₂Ph),4.60-4.57 (0.5H, m, H-3′), 4.56-4.54 (0.5H, m, H-3′), 4.44-4.28 (3H, m,H-5′, NHCHCH₃), 4.20-4.17 (1H, m, H-4′), 3.33 (6H, q, J=7.0 Hz,N(CH₂CH₃)₃), 1.47-1.41 (3H, m, NHCHCH₃), 1.31 (9H, t, J=7.5 Hz,N(CH₂CH₃)₃).

C₃₀H₃₀ClFN₆₀₉P₂S mass required m/z 767.06; found MS (ES⁻) m/z 766.12([M—H]).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN from 90/10 to 0/100 in30 min, F=1 mL/min, λ=254 nm, one peak for two diastereoisomers witht_(R)=16.39 min.

Example 27—In Vitro Cytotoxicity Analyses

A subset of compounds of the invention were assayed for their cytotoxicactivity in an array of different solid tumours and haematologicalmalignancies using the following assay.

Solid tumour and haematological malignancy assay.

In vitro viability assay were performed to assess the effects ofcompounds on cell viability in selected cell lines over 72 h using theCellTiterGlo (CTG, Promega-G7573) assay. The tests were performed induplicates with treatment of compounds at 9 points, 3.16 folds titrationin 96 well plates over −72 h. The compound starting concentrations were198 μM. Cell viability assay using CellTiterGlo in 96-well plate wereperformed. Compound treatment 72 h, standard growth conditions,duplicates. Compounds were dissolved to 40 mM with thawed 100%.Compounds were serially diluted at 3.16 fold in thawed DMSO, and warmedto 37° C. before being dissolved in media (2 μL+200 μL). After compoundswere dissolved in media, media containing compounds were warmed to 37°C. in incubator and then compounds in media were added to cell plates(50 μL+50 μL) in duplicates. The compounds' final concentrations werefrom 198 μM to 19.8 nM. All compound solubilities were checked andrecorded again, then the plates were transferred to CO₂ tissue cultureincubator immediately and incubated for 3 days. DMSO final concentrationis 0.5%. The parent nucleoside in each case was tested as a comparator,as was an exemplary ProTide. The drugs of the ProTide class that weretested were as follows:

The following cell lines were tested and are referred to in the Table 1below:

TABLE 1 Cell line Malignancy Cell line Malignancy MOLT-4 Acute T MCF-7Breast lymphoblastic adenocarcinoma leukaemia CCRF-CEM Acute HL-60Promyelocytic lymphoblastic leukaemia leukaemia RL Non-Hodgkin’s MV4-11Biphenotypic B lymphoma myelomonocytic leukaemia RPMI-8226 Humanmultiple HepG2 Hepatocellular myeloma carcinoma K562 Chronic HT29 Colonmyelogenous adenocarcinoma leukaemia OVCAR-3 Ovarian Mia-Pa-Ca-2Pancreatic adenocarcinoma carcinoma KG-1 Acute myelogenous leukaemia

The results of this screening are presented in Tables 2, 3 and 4.

In Table 3, A represents an EC₅₀ of no more than 0.2 μm; B represents anEC₅₀ of greater than 0.2 μm but no more than 2 μm; C represents an EC₅₀of greater than 2 μm but no more than 5 μm; D represents an EC₅₀ ofgreater than 5 μm but no more than 10 μm; and E represents an EC₅₀ ofgreater than 10 μm. No entry means that screening of the indicatedcompound against the indicated cell line was not carried out.

In Table 4, A represents an EC₅₀ of no more than 0.2 μm; B represents anEC₅₀ of greater than 0.2 μm but no more than 1 μm; C represents an EC₅₀of greater than 1 μm but no more than 5 μm; D represents an EC₅₀ ofgreater than 5 μm but no more than 10 μm; and E represents an EC₅₀ ofgreater than 10 μm. Letters in bold represent an EC₅₀ less than that ofthe exemplary ProTide A.

In Tables 2, 3 and 4, ‘Ab EC₅₀ ‘refers to the absolute EC₅₀ and ‘Topinhibit’ refers to the percentage inhibition achieved at the highestconcentration tested.

TABLE 2 Mia-Pa-Ca-2 HT29 HepG2 OVCAR 3 MCF-7 Ab Top Ab Top Ab Top Ab TopAb Top EC₅₀ Inhibit EC₅₀ Inhibit EC₅₀ Inhibit EC₅₀ Inhibit EC₅₀ InhibitCompound (μM) (%) (μM) (%) (μM) (%) (μM) (%) (μM) (%) Gemcitabine 0.0379 0.22 65 28.0 54 >198 44 0.011 53 8 0.07 79 0.48 62 83.13 52 >198 48.50.07 52 B 0.53 82 8.55 62 21.33 57 >198 33.5 2.71 54 FUDR 0.35 90 0.2282 84.02 61 2.32 66 7.23 73 4 0.11 67 0.25 77 >198 48 2.81 67.5 15.80 66C 1.01 89 1.91 81 70.60 65 — — 27.25 89 8-CI- 0.42 85 1.96 73 — — — —1.83 85.7 Adenosine 5 0.43 87.6 1.41 76.5 — — — — 1.69 88 D 1.97 99.73.82 94 — — — — 4.30 99.6 Cordycepin 84.73 88 32.74 87 — — — — — — 73.32 98.90 7.79 79 — — — — — — 6 40.89 95.7 24.34 82 — — — — — — E 7.9298 10.75 97 — — — — — — RPMI- CCRF-CEM MOLT-4 K562 HL-60 RL 8226 Ab TopAb Top Ab Top Ab Top Ab Top Ab Top EC₅₀ Inh EC₅₀ Inh EC₅₀ Inh EC₅₀ InhEC₅₀ Inh EC₅₀ Inh Compound (μM) (%) (μM) (%) (μM) (%) (μM) (%) (μM) (%)(μM) (%) Gemcitabine 0.02 101 0.004 88 — — — — — — — — 8 0.04 99.9 0.00984 >198 29 0.08 98 0.20 66.5 — — B — — 0.24 76.4 — — — — — — — — FUDR0.04 91.9 — — — — — — — — 0.11 99 4 0.03 97.1 — — — — — — — — 0.05 95 C— — — — — — — — — — — — 8-CI- — — — — — — — — — — — — Adenosine 5 — — —— — — — — — — — — D — — — — — — — — — — — — Cordycepin — — 100.65 76.5117.51 94 117.58 56 >198 16.6 >198 17 7 — — 25.30 90 9.16 94 6.97 10011.10 93 1.60 98 6 — — 14.19 95 52.28 81 62.50 80 88.81 72 — — E — —5.23 96 10.95 87 31.13 97 18.29 90.5 19.89 97.8

TABLE 3 HT29 HepG2 OVCAR 3 MCF-7 Ab Top Ab Top Ab Top Ab Top EC₅₀Inhibit EC₅₀ Inhibit EC₅₀ Inhibit EC₅₀ Inhibit Compound (μM) (%) (μM)(%) (μM) (%) (μM) (%) Clofarabine B 64 C 64 E 48 B 55 1 B 66 — — E — B59 2 B 62 — — E 48 B 54 3 B 61 C 53 E 49 C 59 A C 94 — — — — D 99.6RPMI- CCRF-CEM MOLT-4 K562 HL-60 RL 8226 Ab Top Ab Top Ab Top Ab Top AbTop Ab Top EC₅₀ Inh EC₅₀ Inh EC₅₀ Inh EC₅₀ Inh EC₅₀ Inh EC₅₀ InhCompound (μM) (%) (μM) (%) (μM) (%) (μM) (%) (μM) (%) (μM) (%)Clofarabine A 98.7 A 84.1 E 23 A 97 B 77 D 89 1 A 98.7 A 82.7 E 24 A 97B 70 D 91 2 A 98.7 A 98 E 29 A 98 B 65 D 92 3 A 99.6 A 96 E 24 A 97 B 63D 91.6 A A 99.4 B 93 E 32 B 97 C 99.8 C 97

TABLE 4 HT29 HepG2 MCF-7 MOLT-4 Ab Ab Ab Ab EC₅₀ Top EC₅₀ Top EC₅₀ TopEC₅₀ Top Compound (μM) Inhibit % (μM) Inhibit % (μM) Inhibit % (μM)Inhibit % Clofarabine B 68 C 57 B 54 A 94  A C 86 D 99 D 99 B 100  9 B70 B 53 — 45 A 92 10 B 70 C 55 E 51 A 93 11 B 69 C 58 E 57 A 91 12 0.3771 0.29 54 — 44 0.04 91 13 B 68 — 49 — 49 A 89 14 B 71 B 52 — 48 A 95 150.33 69 0.37 51 — 47 0.03 90 16 B 71 B 53 — 47 A 97 17 B 69 B 52 — 46 A88 18 B 66 B 53 — 44 A 88 19 B 68 B 51 — 47 A 90 20 B 69 B 54 — 48 A 8921 B 69 B 53 — 49 A 90 22 B 69 — 47 — 50 A 89 23 B 68 C 51 — 48 A 89 24B 70 C 54 — 49 A 88 25 B 67 — 49 — 43 A 86 26 B 64 — 49 — 44 A 84 OVCAR3 CCRF-CEM K 562 HL-60 Ab Ab Ab Ab EC₅₀ Top EC₅₀ Top EC₅₀ Top EC₅₀ TopCompound (μM) Inhibit % (μM) Inhibit % (μM) Inhibit % (μM) Inhibit %Clofarabine E 42 A 99 E 32 A 97  A E 94 A 99 E 85 B 97  9 E 43 A 99 E 40A 96 10 E 42 A 99 E 37 A 96 11 E 70 A 99 E 50 A 96 12 >198 41 <0.0299 >198 40 0.07 96 13 E 80 A 99 E 44 A 96 14 E 43 A 99 E 36 A 96 15 >19841 <0.02 99 >198 36 0.06 96 16 E 44 A 99 E 33 A 96 17 E 47 A 99 E 37 A96 18 E 47 A 99 E 40 A 96 19 E 51 A 99 E 37 A 95 20 E 49 A 99 E 35 A 9621 E 47 A 99 E 32 A 96 22 E 73 A 99 E 62 A 95 23 E 63 A 99 E 35 A 96 24E 67 A 99 E 49 A 95 25 E 24 A 99 E 33 A 95 26 E 62 A 99 E 69 A 95 KG-1RL RPMI-8226 Ab Ab Ab EC₅₀ Top EC₅₀ Top EC₅₀ Top Compound (μM) Inhibit %(μM) Inhibit % (μM) Inhibit % Clofarabine A 93 B 61 E 80  A B 93 C 90 C94  9 A 92 B 53 E 81 10 A 92 — 48 E 80 11 A 92 — 47 E 81 12 0.14 91 — 4739.55 79 13 A 93 — 48 E 88 14 A 93 — 49 E 78 15 0.09 92 — 49 30.28 80 16A 93 — 49 E 84 17 A 92 — 48 E 82 18 A 91 — 48 E 84 19 A 92 B 57 E 79 20A 92 B 69 E 80 21 A 92 C 70 E 81 22 A 92 — 42 C 86 23 A 93 — 39 E 85 24A 91 — 39 E 86 25 A 91 — 44 C 83 26 A 91 E 56 C 93

As can be seen from Tables 3 and 4, certain compounds of the inventionwere more active than the ProTide A against a range of different cancercell lines.

Example 28—Metabolic Stability

The assay was performed according to the published procedure (Kuhnz, W.;Gieschen, H. Drug Metab. Dispos. 1998, 26, 1120-1127).

Pooled cryopreserved hepatocytes were thawed, washed, and re-suspendedin Krebs-Henseleit buffer (pH 7.3). The reaction was initiated by addingthe test compound (1 μM final concentration) into cell suspension andincubated in a final volume of 100 μL on a flat-bottom 96-well plate for0 and 120 min, respectively, at 37° C./5% CO₂. The reaction was stoppedby adding 100 μL of acetonitrile into the incubation mixture. Sampleswere then mixed gently and briefly on a plate shaker, transferredcompletely to a 0.8 mL V-bottom 96-well plate, and centrifuged at 2500×gfor 15 min at room temperature. Each supernatant (150 μL) wastransferred to a clean cluster tube, followed by HPLC-MS/MS analysis ona Thermo Electron triplequadrupole system.

For comparison, the parent nucleoside and a corresponding ProTide weretested in the same assay

The results are shown in Tables 5 and 6:

TABLE 5 Human hepatocytes Compound Mean half-life (min.) D  53 5  188-Cl-Adenosine >120  B 139 8  27 Gemcitabine — C  24 4  15 FUDR  69 E 90 7  27 Cordycepin  48

TABLE 6 Human hepatocytes Compound Mean half-life (min.) A  24 1 In therange 20 to 100 2 In the range 20 to 100 3 In the range 20 to 100Clofarabine >120

Example 29—Evaluation of clofarabine and selected clofarabinediphosphate phosphoramidates in a KG1a cell line model of acute myeloidleukaemia (AML)

SUMMARY

Clofarabine and Examples 1 and 2 were selected for analysis to determinewhether: (1) the compounds of the invention possessed increased potencywhen compared to the parental compound and (2) the compounds of theinvention preferentially targeted leukaemic stem cells (LSCs). In orderto establish this, the acute myeloid leukaemia cell line, KG1a, wasemployed as it manifests a minor stem cell-like compartment with adistinct immunophenotype (Lin⁻/CD34⁺/CD38⁻/CD123⁺). The compounds wereevaluated over an extended dose range. In addition, the effects of thecompounds on the stem cell compartment were evaluated over the entiredose range. The mean clofarabine LD₅₀ (the concentration of drugrequired to kill 50% of the cells) was 1.69×10⁻⁸M. Example 1 showed asimilar mean LD₅₀ value (1.37×10⁻⁸M), whereas Example 2 showedsignificantly increased mean LD₅₀ values (4.38×10⁻⁸M and 7.10×10⁻⁸Mrespectively).

Under normoxic conditions, the LSC (Lin⁻/CD34⁺/CD38⁻/CD123⁺) compartmentconstituted ˜3.5% of the total cell line. However, under hypoxicconditions (1% and 5% oxygen) the proportion of LSCs showed atime-dependent increase (up to 23.7%). Furthermore, this was reversedwhen the cell line was transferred back into normoxic conditions. Therewas no significant difference in the fraction of LSCs in the culturesbetween 1% and 5% oxygen. When considering the relative potency of thecompounds under normoxic and hypoxic conditions (5% oxygen), clofarabineshowed a significant increase in mean LD₅₀ under hypoxic conditions(1.69×10⁻⁸M to 5.3 1×10⁻⁸M; P=0.01). In contrast, Example 1 showed nosignificant difference in mean LD₅₀ values.

Objectives

1. To generate a complete cytotoxicity dose-response curve for a seriesof selected compounds and their respective parental nucleoside in KG1acells

2. To establish the effects of the tested compounds on the LSCcompartment over the entire range of concentrations used

3. To compare the effects of the tested compounds under hypoxicconditions (1% and 5% oxygen)

4. To investigate the persistence of γH2A.X foci in LSCs and bulk tumourcells following treatment with clofarabine and the tested compoundsunder hypoxic conditions

Materials and Methods

KG1a Cell Culture Conditions

The acute myeloid leukaemia (AML) KG1a cell line was maintained in RPMImedium (Invitrogen, Paisley, UK) supplemented with 100 units/mLpenicillin, 100 μg/mL streptomycin and 20% foetal calf serum. Cells weresubsequently aliquoted (10⁵ cells/100 μL) into 96-well plates and wereincubated at 37° C. in a humidified normoxic (20% oxygen) or hypoxic (1%or 5% oxygen) atmosphere for 48h in the presence of clofarabine, orclofarabine diphosphate phosphoramidates of the invention (1×10⁻¹° M−1×10⁻⁶M). In addition, control cultures were carried out to which nodrug was added. Cells were subsequently harvested by centrifugation andwere analysed by flow cytometry using the Annexin V assay.

Measurement of In Vitro Apoptosis

Cultured cells were harvested by centrifugation and then resuspended in1954, of calcium-rich buffer. Subsequently, 5 μL of Annexin V (CaltagMedsystems, Botolph Claydon, UK) was added to the cell suspension andcells were incubated in the dark for 10 mins prior to washing. Cellswere finally resuspended in 190 μL of calcium-rich buffer together with10 μL of propidium iodide. Apoptosis was assessed by dual-colourimmunofluorescent flow cytometry as described previously. SubsequentlyLD₅₀ values (the dose required to kill 50% of the cells in a culture)were calculated for each nucleoside analogue and diphosphatephosphoramidate.

Immunophenotypic Identification of the Leukaemic Stem Cell Compartment

KG1a cells were cultured for 48h in the presence of a wide range ofconcentrations of each nucleoside analogue and their respectivediphosphate phosphramidate. Cells were then harvested and labelled witha cocktail of anti-lineage antibodies (PE-cy7), anti-CD34 (FITC),anti-CD38 (PE) and anti-CD123 (PERCP cy5). The sub-population expressinga leukaemic stem cell (LSC) phenotype were subsequently identified andwere expressed as a percentage of all viable cells left in the culture.The percentages of stem cells remaining were then plotted on adose-response graph and the effects of the diphosphate phosphoramidatewere compared with the parental nucleosides.

Cell Sorting

KG1a cells were grown under hypoxic conditions for 7 days in order togenerate 10⁷ cells and increase the percentage of cells expressing anLSC phenotype. Subsequently, Lin⁻/CD34⁻/CD38⁻/CD123⁺LSCs andLin⁻/CD34⁺/CD38^(+/CD)123⁺‘bulk’ tumour cells were purified by highspeed cell sorting using a FACS Melody cell sorter (Becton Dickenson)and were placed back into hypoxic cell culture conditions prior to theaddition of clofarabine or clofarabine diphosphate phosphoramidates.

γH2A.X Phosphorylation Assay

Phosphorylation of the histone variant γH2A.X occurs as a rapid responseto double strand DNA breaks. The γH2A.X Phosphorylation Assay Kit (Flowcytometry) is a cell-based assay formatted for flow cytometric detectionof levels of phosphorylated Histone γH2A.X (Merck, UK). LSC and ‘bulktumour’ cells were cultured in 96-well plates in the presence ofclofarabine or clofarabine diphosphate phosphoramidates. After 2h ofexposure to drug, the cells were harvested by centrifugation and thenfixed and permeabilised in preparation for staining and detection.Histone γH2A.X phosphorylated at serine 139 is detected by the additionof the anti-phospho-Histone γH2A.X conjugated to APC. Cells were thenrun on a flow cytometer to quantitate the number of cells stainingpositive for phosphorylated Histone γH2A.X.

Statistical Analysis

The data obtained in these experiments were evaluated using one-wayANOVA. All data was confirmed as Gaussian or a Gaussian approximationusing the omnibus K2 test. LD₅₀ values were calculated from thenon-linear regression and line of best-fit analysis of the sigmoidaldose-response curves. All statistical analyses were performed usingGraphpad Prism 6.0 software (Graphpad Software Inc., San Diego, Calif.).

Results

In Vitro Cytotoxicity Assay

The in vitro drug sensitivity of KG1a cells was assessed using theAnnexin V/propidium iodide assay. The sigmoidal dose-response curves foreach compound tested are shown in FIG. 1A-C. The results are summarisedin Table 7.

TABLE 7 Compound Mean LD₅₀ (nM) Clofarabine 16.9 Example 1 13.7 Example2 71.0

Clofarabine and the compounds of the invention showed potency in thenanomolar range; clofarabine and Example 1 had similar LD₅₀ values withExample 1 being slightly more potent.

The Fraction of KG1a Cells Expressing an LSC Phenotype is Modulated byHypoxia

KG1a cells were grown under normoxic and hypoxic conditions and the LSCphenotype was monitored over time. Under normoxic conditions, the LSCphenotype was stably maintained in approximately 3.5% of the cells inculture. In contrast, under hypoxic conditions, the fraction of thecells in culture expressing the LSC phenotype increased in atime-dependent manner (FIG. 3A). When cells were then transferred backinto normoxic culture conditions, the fraction of cells expressing theLSC phenotype returned to −3.5%, again in a time-dependent manner (FIG.3B).

Comparison of Clofarabine and Compounds of the Invention Under Normoxicand Hypoxic Conditions

Given the observed increase in an LSC phenotype under hypoxicconditions, the relative potency of clofarabine and the compounds wasassessed in KG1a cells cultured under normoxic and hypoxic (5% 02)conditions. FIG. 4 shows the overlaid sigmoidal dose-response curves forclofarabine and the compounds of the invention. The results aresummarised in Table 8:

Normoxia Hypoxia Ratio Mean Mean Normoxia/ Compound LD₅₀ (nM) LD₅₀ (nM)Hypoxia Clofarabine 16.9 53.1 31.8 Example 1 13.7 16.3 84.0 Example 271.0 95.0 74.7

As can be seen, clofarabine showed a significant reduction in potencywhen used on cells cultured under hypoxic conditions. This reduction inpotency was not observed when Examples 1 and 2 were used under the samehypoxic conditions.

Clofarabine Induces Significantly Less DNA Damage in LSCs than BulkTumour Cells Under Hypoxic Conditions

In an attempt to understand why clofarabine-treated cells were lesssensitive to the effects of the drug under hypoxic conditions, cellswere grown under hypoxic conditions and then the LSC and bulk tumourfractions were purified using high-speed cell sorting. The purifiedcells were then exposed to drug for 2 hours and the amount of DNA damagewas quantified using a γH2A.X phosphorylation assay. FIG. 5 shows thatthe level of DNA damage in the LSCs was significantly lower than thebulk tumour cells following treatment with clofarabine. In contrast,Examples 1 and 2 induced similar levels of DNA damage in both the LSCsand the bulk tumour cells.

-   -   1. In terms of stem cell targeting, all of the compounds tested        showed evidence of stem cell targeting at concentrations above        10⁻⁸M. Example 1 showed a trend towards increased selectivity        against Lin⁻/CD₃₄ ⁺/CD₃₈″/CD₁₂₃ ^(k) LSCs.    -   2. The LSC phenotype was shown to be inducible under hypoxic        conditions and this was reversed when the cells were        reintroduced to normoxic culture conditions. The dynamics of the        changes in LSC fraction suggests a plasticity in the phenotype        rather than a selective expansion/contraction in a fixed LSC        sub-population.    -   3. Clofarabine showed a significant reduction in potency when        used on cells cultured under hypoxic conditions. This reduction        in potency was not observed when Examples 1 and 2 were used        under the same hypoxic conditions. Many cancers exist in a        hypoxic state in the human body.    -   4. One explanation for the reduced clofarabine potency under        hypoxic conditions is that there is a significant expansion of        the LSC fraction under these conditions and LSCs showed        significantly lower DNA damage, as measured by phosphorylation        of γH2A.X, following short-term exposure to clofarabine. In        contrast, Examples 1 and 2 induced similar levels of DNA damage        in the bulk tumour cells and LSCs under the same conditions.

We claim:
 1. A compound of formula (I), or a pharmaceutically acceptablesalt thereof:

wherein R¹ is selected from: C₁-C₂₄-alkyl,C₀-C₄-alkylene-C₃-C₈-cycloalkyl and C₀-C₄-alkylene-aryl; R² and R³ areindependently selected from H, C₁-C₆-alkyl and C₁-C₃-alkylene-R⁷; R⁴ isH; R⁵ is aryl; R⁶ is

R⁷ is selected from aryl, imidazole, indole, SR^(a), OR^(a), CO₂R^(a),CO₂NR^(a)R^(a), NR^(a)R^(b)and NH(=NH)NH₂; Z¹ is 0; X is at eachoccurrence a pharmaceutically acceptable cation; wherein each aryl iseither phenyl or naphthyl; wherein where any of R¹, R², R³, R⁵ or R⁷ isan alkyl, cycloalkyl, or aryl, that alkyl, cycloalkyl, or aryl group isoptionally substituted with from 1 to 4 substituents selected from:halo, nitro, cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a), NR^(a)C(O)R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a), SR^(a), SOR^(a), SO3R^(a),SO₂R^(a), SO₂NR^(a)R^(a), CO₂R^(a), C(O)R^(a), CONR^(a)R^(a),CR^(a)R^(a)NR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl andC₁-C₄-haloalkyl; wherein R^(a) is independently at each occurrenceselected from: H and C₁-C₄-alkyl; and R^(b) is independently at eachoccurrence selected from: H, and C₁-C₄-alkyl and C(O)—C₁-C₄-alkyl. 2.The compound of claim 1, wherein R¹ is selected from C₅-C₇-cycloalkyl,C₁-C₈-alkyl, and benzyl.
 3. The compound of claim 1, wherein R¹ isselected such that it comprises five or more carbon atoms.
 4. Thecompound of claim 2, wherein R¹ is benzyl.
 5. The compound of claim 4,wherein R¹ is unsubstituted benzyl.
 6. The compound of claim 2, whereinR¹ is C₁-C₈-alkyl.
 7. The compound of claim 1, wherein R³ is H
 8. Thecompound of claim 1, wherein R² is selected from C₁-C₆-alkyl andC₁-C₃-alkylene-R⁷.
 9. The compound of claim 8, wherein R² isC₁-C₄-alkyl.
 10. The compound of claim 1, wherein R² is H.
 11. Thecompound of claim 1, wherein R⁵ is phenyl.
 12. The compound of claim 11,wherein R⁵ is unsubstituted phenyl.
 13. The compound of claim 1, whereinR⁵ is naphthyl.
 14. The compound of claim 13, wherein R⁵ is 1-naphthyl.15. The compound of claim 13, wherein R⁵ is unsubstituted naphthyl. 16.The compound of claim 15, wherein R⁵ is unsubstituted 1-naphthyl.
 17. Amethod for treating cancer, comprising administering to a patient inneed thereof a compound of claim 1, wherein the cancer is a leukemia ora lymphoma.
 18. The method of claim 17, wherein the cancer is a leukemiaselected from the group consisting of acute myeloid leukemia (AML),acute lymphoid leukemia (ALL), chronic myeloid leukemia (CIVIL), chroniclymphoid leukemia (CLL) and biphenotypic acute leukemia (BAL).
 19. Themethod of claim 17, wherein the cancer is a lymphoma selected from thegroup consisting of Hodgkin's lymphoma and non-hodgkin's lymphoma.
 20. Apharmaceutical composition comprising the compound of claim 1, and atleast one pharmaceutically acceptable excipient.